Progenitor Cell Differentiation Into Dendritic Cells Requires Cytokine Mediated PKC-IRF Relationships

Abstract Dendritic Cell (DC) differentiation is a complex system involving multiple progenitors with potential to differentiate into a variety of DC subsets. Understanding the mechanisms regulating these differentiation pathways is critical to understanding how defective DCs arise in cancer. Impaired DC differentiation often results in immunosuppressive cells that either hinder immune activation in disease or promote tumor growth and metastasis. We previously established that the serine-threonine kinase Protein Kinase C β isoform II (PKCβII) is required for human DC differentiation from CD34+ progenitor cells and monocytes, and have recently found that murine bone marrow (BM) cells also need it to become fully differentiated and functional DCs. However, the molecular targets of PKCβII in this pathway remain unclear. It is well established that the transcription factors Interferon Regulatory Factors 4 and 8 (IRF4 and IRF8) are also important for DC differentiation. IRF4 is crucial for the development of conventional DCs (mediated by GM-CSF), while IRF8 is crucial for the development of plasmacytoid and CD8α+DCs (mediated by FLT3-L). We hypothesized that a relationship existed between PKCβII and IRF4/8, and investigated the effects of PKC activation on IRF4/8 expression. Using human progenitor cell lines and murine BM cells we found that PKC activation upregulated IRF4 and IRF8 expression, while PKC inhibition downregulated IRF4 and IRF8. PKC inhibition also prevented these cells from differentiating into DCs, as determined by their phenotypic markers, physical characteristics, and T-cell stimulatory activity. However, we found that in progenitor cells GM-CSF (a known PKCβII activator) decreased IRF8 expression while upregulating IRF4 expression. This led us to investigate the differential effects of GM-CSF and FLT3-L on the PKC-IRF relationship. We saw that FLT3-L treatment of murine BM cells caused an upregulation of IRF8 and stimulated DC differentiation, and that DC differentiation and IRF8 upregulation were both lost in the presence of a PKC inhibitor. Using Image Stream analysis we found that FLT3-L treatment of progenitor cells activated PKCβII and PKCα. To determine which PKC(s) mediates the FLT3-L driven upregulation of IRF8, we used PKCα knockout (KO) BM and saw that cells were still able to differentiate into DCs and IRF8 levels were still being upregulated. Thus, PKCβII is the PKC that mediates FLT3-L driven DC differentiation and IRF8 upregulation. To determine what molecules could be acting downstream of PKCβII in regulating IRF4/8, we again used Image Stream analysis and visualized STAT3 and STAT5 translocation into the nucleus. Using murine BM cells we found that STAT3 and IRF8 nuclear localization increased with FLT3-L treatment, while GM-CSF treatment caused increased STAT5 and IRF4 nuclear localization. When looking at human monocytes and the human monocytic progenitor cell lineTHP-1 we saw similar effects: GM-CSF treatment increased STAT5 and IRF4 nuclear localization, while pan-PKC inhibition decreased basal STAT5 and IRF4 nuclear localization. Interestingly, these human monocytes and THP-1 cells had lower nuclear levels of STAT3 and IRF8 following FLT3-L treatment – possibly because these cells are already somewhat committed to the monocyte-derived conventional DC pathway. However, in murine early progenitor cells, after 15 minutes of PKC activation we saw increased STAT3 activation, indicating that PKC-regulated STAT3 activation is playing a role earlier in the differentiation process. To find the progenitor cells immediately effected by PKCβII activation, we used IRF8-eGFP murine BM and saw that PKC activation caused induced IRF8 expression as early as in the multi-potent progenitor cells (MPP2 and MPP3), and this upregulation continued to increase as cells differentiated to CD11b+progenitor cells and GMP. These studies indicate that PKCβII is activated in progenitor cells by either FLT3-L or GM-CSF, causing an upregulation of IRF8 or IRF4, respectively. PKCβII may be acting through STAT5 and STAT3 to induce IRF4 and IRF8, depending on the cytokine treatment. By having a better understanding of how PKCβII regulates the expression of these transcription factors, which are required for DC differentiation, we can manipulate the PKCβII-IRF relationship to drive or impair DC differentiation in pathological settings, and may improve DC-vaccine development. Disclosures: No relevant conflicts of interest to declare.

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GM-CSF Controls Proliferation and Survival of the Granulocyte Lineage through the Transcription Factors STAT5A/B.

Blood ◽

10.1182/blood.v112.11.1272.1272 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1272-1272

Author(s):

Akiko Kimura ◽

Michael A. Rieger ◽

WeiPing Chen ◽

James M. Simmon ◽

Gertraud Robinson ◽

...

Keyword(s):

Transcription Factors ◽

Progenitor Cells ◽

Cell Tracking ◽

Molecular Mechanisms ◽

White Blood Cells ◽

Mutant Mice ◽

Colony Stimulating Factor ◽

Hematopoietic Stem ◽

Gm Csf ◽

Stimulating Factor

Abstract Neutrophils, one kind of granulocytes, are the most abundant type of white blood cells in human peripheral blood and form an integral part of the immune system. In addition, the majority of acute myelogeneous leukemia (AML) cells are from the granulocyte lineage. Granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) control migration, proliferation and survival of granulocytes. G-CSF and GM-CSF activate the transcription factors STAT5A/B (STAT5), which are essential for the development of T and B cells and the erythroid lineage. However, it is not clear to what extent G-CSF or GM-CSF signaling through STAT5 controls the differentiation, proliferation, survival in granulocyte lineage. STAT5 is not only essential for normal development and its constitutive activation has been linked to AML patients with Flt3 mutations. The objective of this study was to explore the contribution of STAT5 in G-CSF- and GM-CSF-induced granulopoiesis and to elucidate the underlying molecular mechanisms. Towards this goal, the Stat5a/b genes were deleted in mouse hematopoietic stem cells in vivo using Cre-loxP-mediated recombination (mutant mice). Injection of 5-FU resulted in a cytokine storm, which in controls, but not in mutant mice, led to a 10-fold elevation of neutrophils. Strikingly, the distribution of myeloid progenitor populations in bone marrow was not altered in STAT5-null animals in homeostasis. Colony assays were performed to address which cytokine controls granulopoiesis from these progenitors. While common multipotent progenitor cells (CMPs) and granulocyte macrophage progenitor cells (GMPs) from control mice formed large colonies in the presence of GM-CSF, mutant cells responded poorly. No difference between control and mutant colonies was observed in the presence of G-CSF. To investigate GM-CSF-mediated survival, apoptosis-assays were performed with peritoneal neutrophils. Greatly elevated apoptosis was observed with STAT5-null neutrophils. To further dissect the contribution of apoptosis and/or proliferation in the observed defects, long-term time-lapse imaging and single cell tracking was applied. Control and STAT5-null GMPs were cultured with GM-CSF and individual cells and all their progeny were continuously observed for 5 generations. Despite an equal number of initial GMPs responding to GM-CSF, the generation time of STAT5-null GMP-derived progeny was significantly prolonged in each generation and the number of cell death events increased dramatically from generation to generation. Therefore, GM-CSF-mediated STAT5 signaling is necessary to generate high numbers of granulocytic cells from GMPs by providing pro-survival and pro-proliferation signals. To identify GM-CSF-mediated and STAT5-dependent genetic cascades that control proliferation and survival of the granulocyte lineage, we performed gene expression profiling and ChIP-seq of control and STAT5-null CMPs, GMPs and neutrophils. STAT5 target genes specific to CMPs, GMPs and neutrophils were identified and their contribution to normal granulopoiesis is currently being investigated.

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Increased avidity for Dpp/BMP2 maintains the proliferation of eye progenitors in Drosophila

10.1101/032995 ◽

2015 ◽

Author(s):

Marta Neto ◽

Fernando Casares

Keyword(s):

Transcription Factors ◽

Progenitor Cells ◽

Progenitor Cell ◽

Normal Development ◽

Local Environment ◽

Proliferative Potential ◽

Normal Organ ◽

Extracellular Signals ◽

Extracellular Matrix Components ◽

Tsh Cells

During normal organ development, the progenitor cell state is transient: it depends on specific combinations of transcription factors and extracellular signals, that cooperate to sustain the proliferative potential and undifferentiated status of organ progenitor cells. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of specific signals from the local environment is often critical to trigger this overgrowth. Therefore, the identification of the specific combinations of transcription factors and signals that promote or oppose proliferation in progenitor cells is essential to understand normal development and disease. We have investigated this issue by asking what signals may promote the proliferation of eye progenitors in Drosophila. Two transcription factors, the MEIS1 homologue homothorax (hth) and the Zn-finger teashirt (tsh) are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. Here we show that Hth+Tsh-induced tissue overgrowth requires the BMP2 ligand Dpp and the activation of its pathway. In Hth+Tsh cells, the Dpp pathway is abnormally hyperactivated. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is maintained use Dpp to enhance their proliferation.

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The Histone Methyltransferase Setd8 Alters the Chromatin Landscape and Regulates the Expression of Key Transcription Factors during Erythroid Differentiation

Blood ◽

10.1182/blood-2018-99-110999 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2568-2568

Author(s):

Jacquelyn Lillis ◽

Jeffrey Malik ◽

Tyler A Couch ◽

Michael Getman ◽

Laurie A. Steiner

Keyword(s):

Transcription Factors ◽

Progenitor Cells ◽

Transcriptional Repression ◽

Conflicts Of Interest ◽

Large Fraction ◽

Erythroid Differentiation ◽

Cell Number ◽

P Value ◽

Histone H4 ◽

And Control

Abstract Setd8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. Setd8 mRNA is expressed ~10-fold higher in erythroid cells than any other cell type (biogps.org) and Setd8 protein levels increase in concert with GATA1 levels during erythroid differentiation of CD34+ HSPCs, suggesting Setd8 may have a role regulating the erythroid transcriptome. Consistent with this hypothesis, erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia and global transcriptomic analyses of sorted populations of E10.5 Sed8 null and control erythroblasts demonstrated a profound defect in transcriptional repression, with 340/345 differentially expressed genes (DEG) expressed at higher levels in the Setd8 null cells than controls (Malik Cell Reports 2017). Primitive erythroblasts mature and enucleate in a semi-synchronous manner in circulation. To better understand the function of Setd8 in regulating the erythroid transcriptome, we extended our transcriptomic analyses by performing RNA-seq in sorted E9.5 Sed8 null (EpoRCre+; Setd8 Δ/Δ) and control (EpoRCre+; Setd8 Δ/+) erythroblasts. The Setd8 null cells failed to repress 20/137 (15%) of the genes that are down regulated in control cells from E9.5 to E10.5. Although relatively few genes were impacted, those genes were enriched for the pathway "Oxidative Stress" (adjusted p-value 0.009) suggesting that Setd8 may regulate specific functions during terminal erythroid maturation. We next compared the DEG in Setd8 null erythroblasts to transcriptomic changes that occur as a cell transcends the hematopoietic hierarchy, gaining lineage specificity while suppressing the multi-lineage transcriptome (GSE14833). A large fraction, 105/345 (~30%), of genes up-regulated in Setd8 null erythroblasts, are also up-regulated in multipotent progenitors compared to proerythroblasts. In contrast, only 16/345 (5%) were also up-regulated in granulocyte-monocyte progenitors suggesting that Setd8 does not repress other lineage restricted signatures. Together, these results suggest that Setd8 regulates repression of the multi-lineage transcriptome during erythroid differentiation from multipotent progenitor cells. To gain insights into how Setd8 regulates the erythroid transcriptome, we performed ATAC-seq (Buenrostro Nature Methods 2013) on sorted populations of erythroblasts from E10.5 Sed8 null and control embryos. Cell number for the Setd8 null samples was limited due to anemia, with ~1000 cells used for each replicate. Setd8 and control replicates were aggregated and accessible regions were identified using MACS2. Regions more accessible in Setd8 null cells were identified by computing a log2 ratio between Setd8 null and control samples using deepTools bamCompare. In addition, we utilized ChIPmentation (Schmidl Nature Methods 2015) to assay H3K27me3 occupancy across the genome of WT E10.5 erythroblasts to identify regions of heterochromatin in maturing erythroblasts. Two replicates were performed using 2.5-5x105 cells per assay, and peak called was done using MACS2. A total of 157 genes were identified that had more accessible chromatin in Setd8 null cells and contained an enrichment for H3K27me3 in WT cells suggesting that these genes should be repressed during normal erythropoiesis. Among these were several DEG that were up-regulated in the Setd8 null cells including Hhex, Cd63, and Gata2. Genomic data integration also identified several additional transcriptional regulators that are active in earlier hematopoietic progenitors but typically silenced during erythroid differentiation including Notch1 and Cebpa. Pathway analysis of the 157 genes identified several stemness-related pathways including "Transcriptional regulation of pluripotent stem cells" and "OCT4, SOX2, NANOG repress genes related to differentiation" (adjusted p-values 0.005 and 0.008, respectively). The chromatin regions that were more accessible in the Setd8 null cells were enriched for the DNA binding motifs of the transcription factor ERG (p-value 1-257), SCL (p-value 1e-193), and NRF1 (p-value 1e-101). Taken together, these data suggest that Setd8 works in concert with erythroid transcription factors to repress the transcriptional network in stem and progenitor cells and establish appropriate patterns of gene expression during erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

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Combined Targeting of β-Catenin and FLT-3 Is Synergistically Lethal Against Cultured and Primary AML Blast Progenitor Cells Expressing FLT3 Internal Tandem Duplication (ITD)

Blood ◽

10.1182/blood.v124.21.618.618 ◽

2014 ◽

Vol 124 (21) ◽

pp. 618-618

Author(s):

Saikat Saha ◽

Warren Fiskus ◽

Sunil Sharma ◽

Bhavin Shah ◽

Anna T Rogojina ◽

...

Keyword(s):

Cyclin D1 ◽

Progenitor Cells ◽

Nuclear Localization ◽

Target Genes ◽

Conflicts Of Interest ◽

Adaptor Protein ◽

Beta Catenin ◽

Induced Apoptosis ◽

Nuclear Levels

Abstract β-catenin acts as a co-activator for the T-cell factor (TCF) 4/lymphoid enhancer factor (LEF) 1 bipartite transcription factor at the promoters of the WNT-β-catenin target genes, including cyclin D1, c-Myc and survivin. The canonical WNT-β-catenin pathway is documented to be essential for self-renewal, growth and survival of the AML stem and blast progenitor cells (BPCs), which has also been correlated with a poor prognosis in AML. In AML stem/BPCs expressing mutant FLT3-ITD, increased PI3K/AKT activity causes phosphorylation and inactivation of GSK3β, thereby preventing degradation, promoting stabilization and nuclear localization of β-catenin. Additionally, FLT3 can also directly mediate the tyrosine phosphorylation of β-catenin, thereby stabilizing and promoting the nuclear localization and binding of β-catenin to TCF4. TBL1 (transducin beta-like) is an adaptor protein, which binds to nuclear β-catenin and promotes its co-factor activity with TCF4/LEF1 in mediating transcription of the target genes, including c-Myc, cyclin D1 and survivin. Therefore, we hypothesized that targeted disruption of TBL1-β-catenin binding or depletion of TBL1 would abrogate the pro-growth and oncogenic signaling of β-catenin in AML BPCs, especially those expressing FLT3-ITD. Here, we demonstrate that treatment with 20 to 100 nM of BC2059 (β-Cat Pharmaceuticals), a small molecule, anthraquinone oxime-analog, disrupts the binding of β-catenin to TBL1 (by anti-TBL1 pull down and immunofluorescence analyses) and promotes proteasomal degradation of β-catenin, thereby attenuating the nuclear levels of β-catenin in the cultured (OCI-AML3, MOLM13 and MV4-11), as well as in primary (p) AML BPCs. Concomitantly, BC2059 treatment inhibited the mRNA and protein expression of c-Myc, cyclin D1 and survivin, while de-repressing p21 and Axin2. BC2059 also dose dependently inhibited growth and induced apoptosis of cultured and CD34+ pAML BPCs expressing FLT3-ITD (40 to 60%), but not of normal CD34+ bone marrow progenitor cells (p < 0.01). Transient knockdown of TBL1 or beta catenin (60 to 70%) by lentivirus-transduced shRNA caused loss of viability in MOLM13 cells, which was significantly enhanced by treatment with BC2059 (p < 0.01). BC2059 also induced apoptosis of MOLM13-TKIR cells that were isolated in vitro to exhibit resistance to FLT3 antagonists (approximately 50-fold). Notably, BC2059 treatment (10 mg/kg, t.i.w., by IV injection) also exerted potent in vivo anti-AML activity and significantly improved the survival of immune depleted mice engrafted with cultured and patient-derived pAML BPCs (p < 0.001). Since compared to the control OCI-AML3 cells, BC2059 demonstrated significantly greater lethality against the OCI-AML3 cells ectopically overexpressing FLT3-ITD (approximately 8-fold), we hypothesized that co-treatment with a FLT3 antagonist would further reduce the nuclear levels of β-catenin and enhance the lethal activity of FLT3-antagonist against AML BPCs expressing FLT3-ITD. Indeed, co-treatment with BC2059 (50 nM) and the FLT3-antagonist quizartinib or ponatinib (100 to 200 nM), versus each agent alone, caused more reduction in the nuclear levels and binding of β-catenin to TBL1 (by confocal immunofluorescence analysis). This was associated with greater decline in the expression of c-Myc, cyclin D1 and survivin, but increase in the levels of p21 and BIM. Compared to each agent alone, co-treatment with BC2059 and quizartinib or ponatinib also synergistically induced apoptosis of the FLT3-ITD expressing cultured (MOLM13 and MV4-11) and pAML BPCs (combination indices of < 1.0, by isobologram analyses) but not of normal CD34+ progenitor cells. Treatment with BC2059 (25 to 100 nM) also significantly increased the apoptosis observed by the shRNA mediated incomplete knockdown of TBL1 or β-catenin (approximately 70%) in MOLM13 cells (p < 0.01). Collectively, our findings support that targeted inhibition of the levels and binding of β-catenin to TBL by BC2059 and FLT3-antagonist is a promising approach to exert lethal activity against AML BPCs expressing FLT3-ITD. Further pre-clinical development of this combination therapy against FLT3-ITD expressing AML is progressing. Disclosures No relevant conflicts of interest to declare.

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"C1ORF150", a Novel Mediator of EPO/EPOR/JAK2 Dependent Human Erythroid Progenitor Cell Formation

Blood ◽

10.1182/blood.v128.22.544.544 ◽

2016 ◽

Vol 128 (22) ◽

pp. 544-544

Author(s):

Don M. Wojchowski ◽

Darryl Abbott ◽

Edward Jachimowicz ◽

Matthew Held

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Cell Formation ◽

Refractory Anemia ◽

Red Cell ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cell ◽

Erythroid Progenitor Cells

Abstract An understanding of cellular events that are propagated within erythroid progenitor cells upon HGF-R / JAK complex activation is of basic importance for generating new insight into regulated red cell formation, anemia and myeloproliferative disease. Using the EPO/EPOR/JAK2 system as a paradigm, our group is successfully applying post-translational modification-based proteomics to uncover important new mediators of EPO-dependent human erythropoiesis (certain of which may also relate to EPO's untoward effects on hypertension and cancer progression). Here, we report on the discovery of a novel ORF, "C1ORF150", that is strongly tyrosine phosphorylated in response to EPO, possesses several unique features, and modulates EPO- dependent erythroid progenitor cell formation. In human erythroid progenitor UT7epo cells, EPOR ligation leads to C1ORF150 phosphorylation at tandem tyrosine p-Y69, p-Y89 and p-Y110, p-Y129 sites (up to 10-fold within 15 minutes). For each PTM site, EPOR/JAK2 mediated- phosphorylation was validated in independent LC-MS/MS experiments using Hematide as an EPOR agonist. p-Y69 and p-Y89 are predicted SFK sites, while p-Y110 and p-Y129 are predicted RTK sites (including KIT). Notably, C1ORF150 is conserved in H. sapiens and primates, but is not represented in mouse, rat or lower vertebrates. In addition, C1ORF150 has no obvious orthologues, but within EPO-regulated pY regions exhibits sequence homology with HGAL, an important factor for B cell receptor signaling. To assess C1ORF150's functional effects, we used a lentiviral shRNA loss-of-function approach (80% knockdown efficiency). At physiological EPO levels, the knockdown of C1ORF150 substantially compromised UT7epo erythroid progenitor cell (EPC) survival, including 200% increases in apoptosis observed relative to control sh-NT transduced EPCs (p < 0.01). The ectopic expression of C1ORF150, in contrast, heightened baseline JAK2 activation, and potentiated STAT5 activation following EPO challenge. C1ORF150's subcellular localization proved to be predominantly membrane associated. With regards to expression profiles, C1ORF150 levels were markedly elevated in bone marrow (among 30 human tissues), and during erythroid development were maximal at a CFUe stage. Furthermore, transcriptome profiles of myelodysplastic syndrome (MDS) CD34pos hematopoietic progenitor cells revealed elevated C1ORF150 expression in MDS refractory anemia (p=0.005) and refractory anemia-blast patients (p=0.05) compared to normal controls. In summary, via PTM-proteomics we have discovered "C1ORF150" as a major new pY- regulated EPOR/JAK2 target and membrane associated phosphoprotein that is proposed to have evolved in human erythroid progenitor cells to support EPO's cytoprotective effects, and red cell formation, in part by reinforcing JAK2 and STAT5 activation. In anemia and pre-leukemic contexts, attention also is brought to possible roles for C1ORF150 in the onset and progression of MDS. Disclosures No relevant conflicts of interest to declare.

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Enhanced Generation of Human Hematopoietic Stem/Progenitor Cells From ES and Patient Derived IPS Cells Using a Novel Compound Screen

Blood ◽

10.1182/blood.v118.21.1294.1294 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1294-1294

Author(s):

Roger Emanuel Rönn ◽

Roksana Moraghebi ◽

Carolina Guibentif ◽

Niels-Bjarne Woods

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Ips Cells ◽

Cell Generation ◽

P Value ◽

Hematopoietic Stem ◽

Compound A

Abstract Abstract 1294 The ability to generate hematopoietic stem and progenitor cells from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to identify novel pathways involved in hematopoietic stem and progenitor cell generation and expansion from human ES and iPS cells. By using a small molecule compound library with our optimized iPS-2-blood lineage differentiation protocol, we have identified two novel chemical compounds that specifically enhance the generation of phenotypic adult hematopoietic stem cells. Compound A and compound B both enable increases of CD45/43+CD34+CD38-CD90+CD45RA- cells by 183+/−53%, n=5 and 275%, n=1, respectively. This is in comparison to DMSO carrier control wells, where the percentage of blood cells (CD45/43+) produced per total cells for these experiments was 73+/−2.9%, n=5. The increase in hematopoietic cell output using compound A was highly significant for the adult phenotypical hematopoietic stem cell fraction with a P-value of 0.03, n=5 (see Figure). Hematopoietic progenitor, CD45/43+ CD34+, counts were also slightly increased for compound A and B at 129+/−32%, n=5 and at 211%, n=1, respectively. Interestingly, no statistically significant increase in the number of total blood CD45/43+ cells was detected at the time of harvest, with either compound; at 108% +/− 8.4%, n=5, for compound A, and 164%, n=1, for compound B. In addition to the improvements, as measured by FACS, compounds A and B both increased the numbers of clonogenic progenitors as measured by CFU-assay, allowing for a 241+/−67%, n=4, and 443%, n=1, increase in total hematopoietic colony counts, respectively. These results identify 2 novel compounds with the ability to expand the more primitive fractions of hematopoietic cells with preferential expansion of the most primitive fraction of hematopoietic cells derived from human ES and iPS lines. We are currently performing transplantation experiments with cells generated using these compounds to assess their repopulating potential. Further studies are being performed to investigate the molecular mechanisms of these compounds for hematopoietic stem and progenitor cell generation from ES and iPS cells. Disclosures: No relevant conflicts of interest to declare.

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How Mesp1 makes a move

The Journal of Cell Biology ◽

10.1083/jcb.201604121 ◽

2016 ◽

Vol 213 (4) ◽

pp. 411-413 ◽

Cited By ~ 2

Author(s):

Robert G. Kelly

Keyword(s):

Transcription Factors ◽

Cell Migration ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fate Specification ◽

Cell Biol ◽

Multipotent Progenitor Cells

The transcription factors Mesp1 and Mesp2 have essential roles in the migration and specification of multipotent progenitor cells at the onset of cardiogenesis. Chiapparo et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505082) identify common Mesp functions in fate specification and Mesp1-specific targets controlling the speed and direction of progenitor cell migration.

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Single-Cell Analysis of Lymphoid-Primed Multipotent Progenitors (LMPPs) Reveal Alterations in Lineage Commitment during Aging

Blood ◽

10.1182/blood.v126.23.244.244 ◽

2015 ◽

Vol 126 (23) ◽

pp. 244-244

Author(s):

Sneha Borikar ◽

Vivek Philip ◽

Jennifer J. Trowbridge

Keyword(s):

Single Cell ◽

Progenitor Cells ◽

Cell Fate ◽

Progenitor Cell ◽

Conflicts Of Interest ◽

Single Cells ◽

Differentiation Potential ◽

Increased Risk

Abstract During aging, the hematopoietic compartment undergoes lineage skewing, biased toward myeloid differentiation at the expense of lymphoid differentiation. This skewing clinically presents as impaired adaptive immunity and an increased risk of myeloproliferative disorders. However, little is known of the regulatory mechanisms underlying these changes in differentiation potential due in part to the inadequacy of current analytic techniques to evaluate lineage potency of individual progenitor cells. Recent demonstration that long-lived hematopoietic progenitor cells drive steady-state hematopoiesis has shifted focus onto the progenitor cell compartment to understand clonal dynamics of native hematopoiesis. Here, we critically assess the functional and molecular alterations in the multipotent progenitor cell pool with aging at the single-cell level. We developed novel in vitro and in vivo assays to define the heterogeneity of the LMPP population and test cell-fate potential from single cells. Our results demonstrate, for the first time, distinct, intrinsic lineage potential of single in vitro LMPPs at the cellular and molecular level. We find that clonal alterations in the lymphoid-primed multipotent progenitor (LMPP) compartment contributes to the functional alterations in hematopoiesis observed during aging. Unbiased single-cell transcriptome analysis reveals that true multipotential clones and lymphoid-restricted clones are reduced with aging, while bipotential and myeloid-restricted clones are modestly expanded. Furthermore, myeloid-restricted clones gain myc driver signatures, molecularly identifying clones emerging during aging that are susceptible to transformation. Our study reveals that aging alters the clonal composition of multipotential progenitor cells, directly contributing to the global loss of the lymphoid compartment and increased susceptibility to myeloid transformation. Disclosures No relevant conflicts of interest to declare.

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Genetic Influences Determining Progenitor Cell Mobilization and Leukocytosis Induced by Granulocyte Colony-Stimulating Factor

Blood ◽

10.1182/blood.v89.8.2736 ◽

1997 ◽

Vol 89 (8) ◽

pp. 2736-2744 ◽

Cited By ~ 119

Author(s):

Andrew W. Roberts ◽

Simon Foote ◽

Warren S. Alexander ◽

Clare Scott ◽

Lorraine Robb ◽

...

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Inbred Strains ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Wild Type ◽

Gm Csf ◽

Cell Mobilization ◽

Stimulating Factor

Abstract The mechanisms involved in the mobilization of progenitor cells into the blood by granulocyte colony-stimulating factor (G-CSF ) and other cytokines are poorly understood. To identify important influences on this complex process, in vivo murine models were used. Granulocyte-macrophage colony-stimulating factor (GM-CSF ) transgenic, Max41 transgenic, W/WV, Mpl-null, GM-CSF receptor (β chain)-null mice, wild-type littermate controls, and six inbred strains of mice were injected with 200 μg/kg/d G-CSF for 5 days. Three parameters of response were monitored: white blood cell count (WCC), peripheral blood progenitor cell (PBPC) numbers, and spleen weight. In all genotypes studied, G-CSF induced increases in these three parameters. However, PBPC mobilization in W/WV and Mpl-null mice was only 30% and 9%, respectively, of that observed in wild-type mice. In contrast, perturbations of GM-CSF signalling had no demonstrable effect on in vivo responses to G-CSF. Broad variability was evident between inbred strains for each parameter of the response to G-CSF. A 10-fold range in response was observed for circulating progenitor cell numbers, similar to that observed for normal human subjects receiving G-CSF. The interstrain differences were in the distribution of mature and progenitor cells between peripheral blood, bone marrow, and spleen rather than in the total numbers of these cells in the body. Results of an F2 intercross of low-responding C57BL/6 and intermediate-responding SJL mice indicated that regulation of progenitor cell mobilization is a complex genetic trait, that there is a correlation between this trait and WCC response (r2 = .5), and that this approach may serve as a useful model for the identification of genes involved in the mobilization process.

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Independent regulation of M-CSF and G-CSF gene expression in human monocytes

Blood ◽

10.1182/blood.v71.6.1529.bloodjournal7161529 ◽

1988 ◽

Vol 71 (6) ◽

pp. 1529-1532 ◽

Cited By ~ 1

Author(s):

E Vellenga ◽

A Rambaldi ◽

TJ Ernst ◽

D Ostapovicz ◽

JD Griffin

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Human Blood ◽

Human Monocytes ◽

Blood Monocytes ◽

Colony Stimulating Factors ◽

Interleukin 3 ◽

Gm Csf ◽

Proliferation And Differentiation

The macrophage and granulocyte colony-stimulating factors, M-CSF and G- CSF, act in vitro to induce proliferation and differentiation of monocyte and granulocyte progenitor cells, respectively. We show here that both of these CSFs can be produced by stimulated human blood monocytes, but the M-CSF and G-CSF genes are independently regulated. Recombinant human interleukin-3 (IL-3) and GM-CSF primarily induce expression of the M-CSF gene and secretion of M-CSF, whereas bacterial lipopolysaccharide primarily induces expression of the G-CSF gene and secretion of G-CSF. These results suggest that under different conditions of in vitro stimulation the monocyte secretes factors that could lead selectively to either granulocyte or monocyte production.

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