Generation of a new de novo model of human AML from C-MYC-transduced normal human CD34+ cord blood cells transplanted into NRG-3/GM-CSF/S mice

Ultrastructural studies of human mast cells (HMCs) and basophils (HBs) are reviewed. Sources of HMCs include biopsies of tissue sites and in situ study of excised diseased organs; isolated, partially purified samples from excised organs; and growth-factor-stimulated mast cells that develop de novo in cultures of cord blood cells. Sources of HBs for study include partially purified peripheral blood basophils, basophils in tissue biopsies, and specific growth factor-stimulated basophils arising de novo from cord blood cells. The ultrastructural studies reviewed deal with identity, secretion, vesicles, recovery, and synthesis issues related to the biology of these similar cells.

Download Full-text

Ex vivo culture of human CD34+ cord blood cells with thrombopoietin (TPO) accelerates platelet engraftment in a NOD/SCID mouse model

Experimental Hematology ◽

10.1016/j.exphem.2006.04.009 ◽

2006 ◽

Vol 34 (7) ◽

pp. 943-950 ◽

Cited By ~ 27

Author(s):

Yvette van Hensbergen ◽

Laurus F. Schipper ◽

Anneke Brand ◽

Manon C. Slot ◽

Mick Welling ◽

...

Keyword(s):

Mouse Model ◽

Cord Blood ◽

Blood Cells ◽

Scid Mouse ◽

Ex Vivo ◽

Human Cd34 ◽

Scid Mouse Model ◽

Cord Blood Cells ◽

Ex Vivo Culture

Download Full-text

Generation of Transgenic T Cells from Human CD34+ Cord Blood Cells

Cytokines and Colony Stimulating Factors ◽

10.1385/1-59259-345-3:327 ◽

2003 ◽

pp. 327-340

Author(s):

Bruno Verhasselt ◽

Evelien Naessens ◽

Veronique Stove

Keyword(s):

T Cells ◽

Cord Blood ◽

Blood Cells ◽

Human Cd34 ◽

Cord Blood Cells

Download Full-text

PATTERN OF CYTOKINE EXPRESSION BY RAT LIVER EPITHELIAL CELLS SUPPORTING LONG-TERM CULTURE OF HUMAN CD34+UMBILICAL CORD BLOOD CELLS

Cytokine ◽

10.1006/cyto.1999.0639 ◽

2000 ◽

Vol 12 (7) ◽

pp. 951-959 ◽

Cited By ~ 5

Author(s):

Mickael Rialland ◽

Anne Corlu ◽

Gennady Ilyin ◽

Florian Cabillic ◽

Isabelle Lamy ◽

...

Keyword(s):

Epithelial Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Blood Cells ◽

Human Cd34 ◽

Cord Blood Cells ◽

Umbilical Cord Blood Cells ◽

Rat Liver Epithelial Cells

Download Full-text

The Acetylation of AML1-ETO Is Required for Leukemogenesis.

Blood ◽

10.1182/blood.v116.21.1588.1588 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1588-1588

Author(s):

Lan Wang ◽

Alexander Gural ◽

Fabiana Perna ◽

Xiaojian Sun ◽

Xinyang Zhao ◽

...

Keyword(s):

Gene Expression ◽

Cord Blood ◽

Blood Cells ◽

Target Genes ◽

Biological Effects ◽

Self Renewal ◽

Mouse Leukemia ◽

Human Cd34 ◽

Cd34 Cells ◽

Cord Blood Cells

Abstract Abstract 1588 Transcription factors and histones are similarly modified through acetylation, phosphorylation, ubiquitination and methylation, which impact on the transcriptional regulation of gene expression and various biological processes in normal and malignant hematopoiesis. The t(8;21) associated AML1-ETO fusion protein is found in 40% of the FAB M2 subtype of acute myeloid leukemia, but how the post-translational modification of AML1-ETO affects its leukemogenicity is largely unknown. Here we show that AML1-ETO directly interacts with the lysine acetyltransferase, p300, via the region containing NHR1 domain and that p300 can acetylate two lysine residues in AML1-ETO and AML1-ETO (exon 9a) in human and mouse leukemia cells. To understand the biological effects of AML1-ETO acetylation, we used human CD34+ cord blood cells as a preleukemia model. The maintenance of CD34+ cells by the acetylation defective form of AML1-ETO was 5 fold less than with AML1-ETO (p<0.01) in the liquid culture assay, and unlike the effect of AML1-ETO, the number of the cobble stone area forming cells (CAFC) was not increased by the mutant AML1-ETO in CAFC assay. However, the block in erythroid and myeloid differentiation conferred by AML1-ETO was still seen in the AML1-ETO acetylation mutant transduced human CD34+ cells. We then approved the impact of acetylation on leukemogenicity using the AML1-ETO9a (AE9a) mouse leukemia model. Mice receiving AE9a acetylation mutant transduced fetal liver cells have not developed leukemia by Day 250, whereas all the mice receiving AE9a transduced cells died due to leukemia before Day 160, with a mean survival time of 109 days (p<0.001). These results suggest that the acetylation of AML1-ETO is required not only for its self-renewal promoting effects and but also for the development of acute leukemia. To gain insight into the mechanisms of AML1-ETO acetylation, we performed luciferase assays and found that the AML1-ETO acetylation mutant lost the ability to activate an M-CSFR promoter driven reporter construct. Furthermore, the expression levels of AML1-ETO activated target genes related to self-renewal were not upregulated in AML1-ETO acetylation mutant transduced human CD34+ cells. These results indicated that the acetylation is crucial to AML1-ETO induced transcription activation. We have also been studying the role of the region containing NHR1 domain (245 to 430 aa) in AML1-ETO: deletion of this region abrogated the binding of p300 to AML1-ETO and led to loss of AML1-ETO lysine acetylation. Furthermore, loss of the region containing NHR1 domain abrogated the self-renewal properties of AML1-ETO and the activation of AML1-ETO target genes in human CD34+ cord blood cells, without affecting its differentiation-blocking activity or its ability to repress gene expression. Given the importance of the acetylation of AML1-ETO in its biological effects, we inhibited p300 function, chemically and using RNA interference; this blocked the transcriptional activation of AML1-ETO target genes, and inhibited the growth of AML1-ETO expressing AML cells in both pre-leukemic and leukemia models. All together, we have found that the acetylation of AML1-ETO via p300 is indispensable for its leukemia-promoting activity and for its ability to activate gene expression. Our work suggests that inhibition of p300 function may represent an important new anti-leukemia strategy that targets self-renewing, leukemia-initiating cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Time Course Studies of the Progeny of Barcoded CD34+ Human Cord Blood Cells Generated in Transplanted NOD/SCID-IL2Rγ−/− Mice Unveil the Clonal Dynamics of Short Term, Intermediate Term and Long Term Repopulating Cells

Blood ◽

10.1182/blood.v120.21.28.28 ◽

2012 ◽

Vol 120 (21) ◽

pp. 28-28

Author(s):

Alice M.S. Cheung ◽

Long V. Nguyen ◽

Annaick Carles ◽

Paul H. Miller ◽

Philip A Beer ◽

...

Keyword(s):

Cord Blood ◽

B Cell ◽

Blood Cells ◽

Bone Marrow Cells ◽

Self Renewal ◽

Post Transplant ◽

Human Cd34 ◽

Cord Blood Cells

Abstract Abstract 28 Hematopoietic stem cells (HSC) exhibit heterogeneity in self-renewal and differentiation activity, but the extent to which this is intrinsically determined and extrinsically regulated is still poorly understood. In the mouse, purities of HSCs can now be achieved to allow such questions to be addressed directly. Interestingly, tracking the outputs of large numbers of serial transplantable clones produced from single-cell transplants, or the clonal progenies of vector-marked/barcoded cells indicate the existence in mice of 2 subsets of HSCs with durable self-renewal ability. These 2 subsets are characterized by distinct lineage output programs that are maintained through the many HSC self-renewal divisions required to serially propagate a clone in vivo. To begin to ask whether similar subsets of human HSCs exist, we have created a diverse lentiviral library encoding an estimated >105 different barcode sequences and GFP, and then used this library to track the in vivo clonal outputs of transduced human CD34+ cord blood cells in xenografted mice. For this experiment, CD34+ cells isolated immunomagnetically to a purity of >80% were exposed to virus for 6 hours in the presence of growth factors and then immediately injected intravenously into 2 sublethally irradiated NOD/SCID-IL2Rγ−/− mice (1.2 × 105 cells per mouse; 30% GFP+ cells after 3 days in vitro). Different subsets of human cells were then isolated by FACS from immunostained bone marrow cells aspirated sequentially from the femurs of the mice at intervals from 4–27 weeks post-transplant and the identity, number and size of clones in each established by next generation sequencing of barcoded amplicons derived from each sample. To identify barcodes arising from PCR and sequencing errors and calibrate clone sizes, we included 3 controls of 20, 100 and 500 cells with a known barcode at each datapoint. The data from these controls allowed a threshold of 20 cells per clone to be established with >95% confidence. We then compared the representation of clones among all samples from each mouse to derive the number and size of all clones detected, assuming a mouse contains 2×108 bone marrow cells. This analysis revealed a total of 154 uniquely barcoded clones containing up to 2×108 human hematopoietic cells in the 2 mice (8–30 × 106 in one and 4–165 × 106 in the other at any single time point). Analysis of the representation of each clone over time showed successive waves of repopulation from different clones with lineage output profiles consistent with those obtained by transplanting separate fractions of CD34+ cord blood cells distinguished by their surface phenotypes. Specifically, we detected 50 clones (32% of all clones) that were not sustained at detectable levels beyond 9 weeks post-transplantation and were characterized by robust myeloid differentiation with variable B cell outputs at 4 weeks. Another 30 clones (19%) showed significant but also transient outputs of either or both the myeloid and B cell lineage, disappearing between week 9 and 16 post-transplant. Mature cell output was detected from a total of 74 clones (48%) at the 27 week time point, among which 36 (23%) were not evident during the first 4 months post-transplant. These late-appearing clones were mostly small (contributing up to 3 × 105 total hematopoietic cells at week 27) and made a significantly higher contribution to the total human myeloid population than to the total human B cell population. Notably, the 12 long term clones that showed robust mature cell output detectable in all 3 sites sampled at week 27 when the mice were sacrificed (left leg vs right leg vs pelvis) contained both myeloid and lymphoid cells but with large (>100-fold) variations in their representation in the 3 different sites. This latter finding suggests less trafficking of human cells between sites than expected from parabiotic mouse experiments or substantial differences in the differentiation control exerted in different locations. Additionally, from one of the mice, we obtained the first direct evidence of a large output of human T cells (>9 × 106) that was part of a long term multi-lineage clone detectable at 27 weeks post-transplant. This first use of a barcoding strategy to analyze the clonal dynamics of normal human CD34+ cells with in vivo repopulating activity demonstrates the power of this approach to analyze their lineage outputs and sets the stage for novel applications to expanded and transformed populations. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CD26 Truncation of GM-CSF and IL-3 Alters Their Functional Interactions

Blood ◽

10.1182/blood.v120.21.1240.1240 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1240-1240

Author(s):

Heather A. O'Leary ◽

Charlie Mantel ◽

Scott Cooper ◽

Christina J Walker ◽

Hal E. Broxmeyer

Keyword(s):

Cord Blood ◽

Receptor Binding ◽

Blood Cells ◽

Colony Formation ◽

Full Length ◽

Binding Studies ◽

Gm Csf ◽

Disease States ◽

Cord Blood Cells ◽

Penultimate Proline

Abstract Abstract 1240 CD26 (DPPIV) is a dipeptidyl peptidase that functions by enzymatically cleaving the penultimate proline or alanine of proteins, resulting in functional alterations. The expression and activity of CD26 is relevant in many disease states including obesity and cancer. Modulation of CD26 activity has been shown to increase homing and engraftment of both cord blood and bone marrow cells after transplant indicating the impressive therapeutic potential of CD26 activity altering compounds. Due to its importance in disease states and their subsequent treatments, it is relevant to study how the activity of CD26 alters the functions of the molecules it cleaves, and subsequently their interactions with each other. Mass spectrometry data from our laboratory has shown that CD26 can cleave the penultimate proline of GM-CSF resulting in the truncated form which has blunted signaling and function. Additional data has recently confirmed that CD26 can cleave IL-3 and results in its diminished function as well. Further, and more importantly, since GM-CSF and IL-3 are members of the IL-3 receptor family, and share a common receptor beta chain, we investigated if CD26 truncation of GM-CSF (TGM) or IL-3 (T3) could inhibit the functional activity of the full length (FL) alternate compound (i.e TGM inhibition of FL IL-3 activity or T3 inhibition of FL GM-CSF activity) in the TF-1 cell line and cord blood cells. We determined that both T3 and TGM could inhibit the colony formation induced by either FL GM-CSF or FL IL-3. This inhibition of function correlated with alterations in reactive oxygen species (ROS) levels that mimicked the truncated versions of either GM-CSF or IL-3 even in the presence of the full length molecules. Strikingly, this inhibition of colony formation did not require a 1:1 ratio of the full length to truncated. Rather, approximately 4–10 fold less truncated could be used to efficiently inhibit the colony formation activity of the full length, even across molecules. Interestingly, the ratio of T3 needed to block the full length GM-CSF (1.25ng/ml T3: 10ng/ml FL GM-CSF) was less than the amount of TGM needed to block the full length IL-3 (2.5ng/ml TGM: 10ng/ml FL IL-3) suggesting that T3 is better at blocking FL GM-CSF than TGM is at blocking FL IL-3. However, the ratios of truncated needed to block the function of self FL molecules are identical for both GM-CSF and IL-3 (1.25ng/ml truncated: 10ng/ml FL). Signaling and receptor binding studies were performed for GM-CSF with TF-1 and CD34+cord blood cells, and showed that the truncated GM-CSF inhibited the Stat-5 and JAK2 signaling of FL GM-CSF at less than a 1:1 (10ng/ml FL: 1.25 ng/ml TGM) ratio. Receptor binding studies found that TGM bound to the GM-CSF receptor more efficiently than the FL form but concentrations required to produce 50% maximum inhibition of binding (IC50) is 8-fold lower for TGM compared to FL-GM-CSF, indicating that T-GM-CSF is a better competitor for binding, than is FL-GM-CSF itself suggesting that this may be how TGM is blocking the effects of FL GM-CSF, and potentially IL-3, in our model. Finally, cells treated with TGM had diminished respiratory and glycolytic rates compared to those treated with full-length cytokine. These data provide the first evidence of relevant interactions, with functional consequences, of the importance of full length and CD26 truncated cytokines across molecules. Disclosures: Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy.

Download Full-text