Induced Pluripotent Stem Cells to Model Blood Diseases

Abstract Our group is developing induced pluripotent stem cell (iPSC) models of myeloid malignancies, including MDS, MPN and AML. We are generating iPSCs from the bone marrow or blood of patients, which can be maintained indefinitely as pluripotent cell lines and, upon in vitro differentiation along hematopoietic lineages, exhibit hallmark features of these diseases. By integrating mutational analyses with cell reprogramming we can derive iPSCs capturing dominant clones, subclones and normal cells from the same patient and thus have established a collection of iPSC lines representing distinct disease stages along the spectrum of myeloid transformation: predisposition syndromes/preleukemic cells/clonal hematopoiesis; low risk MDS; high risk MDS; and MDS/AML. In parallel, we are using the CRISPR/Cas9 system to introduce or correct mutations in normal or malignant iPSCs, respectively, in isogenic settings and sequential CRISPR gene editing to model mutational cooperation. We recently reported that iPSC lines derived from patients with AML re-establish upon differentiation a leukemic phenotype characterized by extensive proliferation of immature myeloid cells that serially transplant a lethal leukemia into NSG mice (Kotini et al. Cell Stem Cell 2017). Strikingly, we observed that the AML-iPSC-derived hematopoietic stem/progenitor cells (HSPCs) contain two morphologically and immunophenotypically distinct cell subpopulations: a cell fraction (adherent, A) exhibiting adherent growth and containing immature cells with an HSC immunophenotype (CD34+/CD38-/CD90+/CD45RA-/CD49f+); and a non-adherent fraction (suspension, S) of more differentiated cells. Fate-tracking experiments revealed a hierarchical organization, with the A cells renewing themselves and continuously giving rise to the S cells through symmetric and asymmetric divisions. The NSG engraftment potential was largely contained within the adherent cell fraction. Thus, AML-iPSCs exhibit the hallmarks of a leukemia stem cell (LSC) model, namely phenotypic and functional heterogeneity and hierarchical organization, with the A fraction containing LSCs that serially transplant leukemia and give rise to more differentiated cells (S fraction) without engraftment potential. LSCs are believed to be a prominent source of AML relapse, but their rarity and the unavailability of universal and specific immunophenotypic markers prohibits their prospective isolation and makes the study of their properties challenging. This new iPSC-based AML-LSC model enables us for the first time to prospectively obtain large numbers of genetically clonal human LSCs and perform genome-wide integrative molecular analyses and large-scale screening to identify key molecular mechanisms sustaining the properties of LSCs as potential new therapeutic targets. Using this model we characterized the effects of previously proposed compounds with LSC selectivity in self-renewal vs differentiation of LSCs. We also screened a small molecule library of 1280 compounds in the A and S cells in parallel to identify compounds with selectivity for the former as candidates for LSC-specific targeting. Disclosures No relevant conflicts of interest to declare.

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JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Blood ◽

10.1182/blood.v128.22.2653.2653 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2653-2653 ◽

Cited By ~ 1

Author(s):

Yuichiro Nakata ◽

Norimasa Yamasaki ◽

Takeshi Ueda ◽

Kenichiro Ikeda ◽

Akiko Nagamachi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Epigenetic Mark ◽

M2 Macrophage ◽

Leukemic Stem Cells ◽

Cell Potential ◽

Hematopoietic Stem

Abstract Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive. To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs. We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. Disclosures No relevant conflicts of interest to declare.

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Inhibition of Lysine-Specific Demethylase 1A (LSD1) Supports Human HSCs in Culture By Preserving Their Immature State

Blood ◽

10.1182/blood-2018-194 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 194-194

Author(s):

Agatheeswaran Subramaniam ◽

Mehrnaz Safaee Talkhoncheh ◽

Kristijonas Zemaitis ◽

Shubhranshu Debnath ◽

Jun Chen ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Stem Cell ◽

Cell Division ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Enrichment Score ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract The molecular mechanisms that govern hematopoietic stem cell (HSC) fate decisions remain incompletely defined. It has been a long-standing goal in the field to gain a better understanding of the genes and pathways that regulate the self-renewal ability of HSCs in order to develop optimal culture conditions in which HSCs can be expanded for clinical benefit. Lysine-specific histone demethylase 1A (LSD1), also known as lysine (K)-specific demethylase 1A (KDM1A), regulates gene expression by specifically eliminating di- and mono-methyl groups on H3 lysine K4 and K9 residues. Studies in mice have shown that, conditional knockdown of LSD1 results in an expansion of bone marrow hematopoietic stem and progenitor cells (HSPCs). However, a complete knockout of LSD1 results in pancytopenia and a dramatic reduction of HSPCs. In this study, we asked whether inhibition of LSD1 would improve the maintenance or expansion of cultured human HSCs derived from umbilical cord blood (UCB). To evaluate the effect of LSD1 inhibition we treated UCB CD34+ cells with three different LSD1 inhibitors (2-PCPA, GSK-LSD1 and RN1) at their respective IC50 values (20µM, 16nM and 70nM) and expanded the cultures for 6 days in serum free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L). Since we (Subramaniam et. al. Haematologica 2018) and others recently have shown that EPCR is a reliable cell surface marker to track UCB derived HSCs during in vitro culture, we quantified the numbers of CD34+EPCR+ cells using flow cytometry and compared to DMSO treated control cultures. Remarkably, treatment with either 2-PCPA or GSK-LSD1 resulted in a more than 10-fold increase of CD34+EPCR+ cells, compared to controls. Further, from dose response experiments we found that 2-PCPA at 1.25 µM expanded the total CD34+ cell population more efficiently than GSK-LSD1, and we therefore used 2-PCPA at this concentration for the subsequent experiments. Using carboxyfluorescein succinimidyl ester (CFSE) labeling to monitor cell division, we found that 2-PCPA did not significantly alter the cell division rate of the cultured CD34+ cells compared to DMSO controls, suggesting that the expansion of CD34+EPCR+ cells is not due to increased proliferation, and that LSD1 inhibition rather may prevent differentiation of the immature HSPCs. To further explore this, we mapped the early transcriptional changes triggered by 2-PCPA in HSCs using gene expression profiling of CD34+CD38-CD45RA-CD90+ cells following 24 hours of culture with or without 2-PCPA treatment. We found that gene sets corresponding to UCB and fetal liver HSCs were significantly enriched upon 2-PCPA treatment compared to DMSO control (Normalized Enrichment Score (NES)=1.49, q=0.05). This suggest that 2-PCPA indeed restricts differentiation and preserves the HSC state upon ex vivo culture. Strikingly, the gene signature induced by LSD1 inhibition was highly similar to that induced by the known HSC expanding compound UM171 (NES=1.43, q=0.11). UM171 is a molecule with unknown target and has also been shown to dramatically expand the EPCR+ population in culture. Finally, the frequency of functional HSCs in DMSO and 2-PCPA treated cultures were measured using limiting dilution analysis (LDA). LDA was performed by transplanting 4 doses (day 0 equivalents of 20000, 1000, 300 and 100 CD34+ cells) of DMSO and 2-PCPA treated cultures into sub lethally irradiated (300cGy) NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human CD45+ cell engraftment in the bone marrow was analyzed 18 weeks' post transplantation. Cultures treated with 2-PCPA showed a 5-fold higher content of long-term repopulating cells per day 0 CD34+ cell equivalent compared to the DMSO control (1 in 615 vs 1 in 3041, p=0.03). Thus, the 2-PCPA treated cultures had significantly enhanced HSCs numbers. To determine the absolute expansion rate, we are currently performing LDA using uncultured cells as well. Altogether our data suggest that LSD1 inhibition supports both phenotypic and functional HSCs in culture by preserving the immature state. Currently we are exploring the possibilities of using LSD1 inhibitors in combination with other known modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

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Enforcing Post-Transcriptional Circuitries to Achieve Human Hematopoietic Stem Cell Expansion

Blood ◽

10.1182/blood.v130.suppl_1.sci-46.sci-46 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. SCI-46-SCI-46

Author(s):

Kristin Hope

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Rna Binding ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Self Renewal ◽

Control Of Gene Expression ◽

Hematopoietic Stem

Abstract The balance between hematopoietic stem cell (HSC) differentiation and self-renewal is central to clinical regenerative paradigms. Unravelling the precise molecular mechanisms that govern HSC fate choices will thus have far reaching consequences for the development of effective therapies for hematopoietic and immunological disorders. There is an emerging recognition that beyond transcription, HSC homeostasis is subject to post-transcriptional control by RNA-binding proteins (RBPs) that ensure precise control of gene expression by modulating mRNA splicing, polyadenylation, localization, degradation or translation. RBPs can synchronously regulate the fates of operationally similar RNAs, in what have been termed RNA regulons. We have used a variety of functional approaches, in combination with unbiased genome- and proteome-scale, methods to define the tenets that govern this regulation and to determine key downstream circuitries of stem cell-regulating RBPs whose targeting could provide the basis for novel regenerative treatments. Through loss-of-function efforts, we have identified the RBP, MSI2, as a required factor for human HSC maintenance. By contrast, at supraphysiological levels, MSI2 has a profound positive effect on human HSC self-renewal decisions. Using a combination of global profiling, including mapping MSI2's targets through cross-linking immunoprecipitation (CLIP)-seq, we show that MSI2 achieves its ex vivo self-renewal-promoting effects by directing a co-ordinated post-transcriptional repression of key targets within the aryl hydrocarbon receptor (AHR) pathway. We are currently exploring the "rules" by which MSI2 influences its downstream effects on target RNAs and how it functions, in combination with other protein interactors, to instill a putative RBP regulatory code in HSCs. HSCs exhibit highly unique epigenomes, transcriptomes and proteomes and it is this distinctive molecular landscape that provides the canvas upon which MSI2, and indeed any other HSC-specific RBP exert their post-transcriptional influence over stem cell function. As such, to decipher the bona fide RNA networks that RBPs function upon in HSCs and to understand how they influence this network to enforce self-renewal, we are working towards performing systematic studies of RBP regulons in these cells specifically. In turn these approaches are elucidating a host of RBPs and post-transcriptional control mechanisms previously unappreciated for their role in HSC control. When modulated appropriately, we can successfully tailor these post-transcriptional regulons to enforce desired HSC outputs ex vivo. In summary, approaches to elucidate key HSC-regulatory RBPs and their protein and RNA interactomes provide valuable insights into a layer of HSC control previously not well understood, and one that can be capitalized on to achieve successful HSC expansion. Disclosures No relevant conflicts of interest to declare.

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EVI1 Is Activated During the Generation of Induced Pluripotent Stem (iPS) Cells.

Blood ◽

10.1182/blood.v114.22.5048.5048 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5048-5048

Author(s):

Leopoldo Laricchia-Robbio ◽

Nuria Montserrat ◽

Alessandra Giorgetti ◽

Juan Carlos Izpisúa Belmonte

Keyword(s):

Stem Cell ◽

Conflicts Of Interest ◽

Integration Site ◽

Ips Cells ◽

The Self ◽

Genomic Locus ◽

Self Renewal ◽

Hematopoietic Stem ◽

Retroviral Integration ◽

Induced Pluripotent

Abstract Abstract 5048 EVI1 gene was first identified as a common site of retroviral integration in murine leukemia models. This gene is part of a complex genomic locus, MDS1-EVI1, that has been described as a target for retroviral integration that may lead to the emergence of a non-malignant dominant hematopoietic stem cell (HSC) clone in mice, in primates, and in humans. These studies suggested that one of the genes encoded by this locus could affect the self-renewal potential of HSC. Recent studies in mice revealed that indeed EVI1 plays an essential role in cell proliferation and it also enhances the self-renewal ability of HSC. The intense attention focused on the MDS1-EVI1 locus as retrovirus integration site prompted us to investigate whether EVI1 might have a role in somatic cell re-programming generated with retroviruses. Recent developments in stem cell research have enabled the re-programming of somatic cells to a pluripotent state using exogenous factors. Induced pluripotent stem (iPS) cells have the potential to differentiate into any cells types and that might be used in the future for clinical therapy. In order to elucidate the molecular events allowing the conversion of adult somatic into pluripotent stem cell, we evaluated EVI1 expression during this process. We found that EVI1 is activated in the early stages of re-programming and then it is silenced once the cells has been fully re-programmed. EVI1 seems to facilitate the initiation of cell re-programming by up-regulating a subset of genes previously described as potent stimulators of stem cells expansion. Disclosures No relevant conflicts of interest to declare.

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Ionizing Radiation Induces Hematopoietic Stem Cell Senescence and Long-Term Bone Marrow Suppression in a p16Ink4a/Arf-Independent manner

Blood ◽

10.1182/blood.v118.21.1345.1345 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1345-1345

Author(s):

Lijian Shao ◽

Wei Feng ◽

Hongliang Li ◽

Yong Wang ◽

Norman Sharpless ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Ionizing Radiation ◽

Hematopoietic Stem Cell ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Sublethal Dose ◽

Independent Manner ◽

Hematopoietic Stem

Abstract Abstract 1345 Many patients receiving chemotherapy and/or ionizing radiation (IR) develop residual (or long-term) bone marrow (BM) injury that can not only limit the success of cancer treatment but also adversely affect their quality of life. Although residual BM injury has been largely attributed to the induction of hematopoietic stem cell (HSC) senescence, neither the molecular mechanisms by which chemotherapy and/or IR induce HSC senescence have been clearly defined, nor has an effective treatment been developed to ameliorate the injury. The Ink4a-Arf locus encodes two important tumor suppressors, p16Ink4a (p16) and Arf. Both of them have been implicated in mediating the induction of cellular senscence in a variety of cells including HSCs. Therefore, we examined the role of p16 and/or Arf in IR-induced HSC senescence and long-term BM suppression using a total body irradiation (TBI) mouse model. The results from our studies show that exposure of wild-type (WT) mice to a sublethal dose (6 Gy) of TBI induces HSC senescence and long-term BM suppression. The induction of HSC senescence is not associated with a reduction in telemore length in HSCs and their progeny, but is associated with significant increases in the production of reactive oxygen species (ROS), the expression of p16 and Arf mRNA, and the activity of senescence-associated β-galacotosidase (SA-β-gal) in HSCs. However, genetical deletion of Ink4a and/or Arf has no effect on TBI-induced HSC senescence, as HSCs from the Ink4a and/or Arf knockout mice after exposure to TBI exhibit similar changes as those seen in the cells from irradiated WT mice in comparison with the cells from un-irradiated mice with correspondent genotypes. In addition, TBI-induced long-term BM suppression is also not attenuated by the deletion of the Ink4a and/or Arf genes. These findings suggest that IR induces HSC senescence and long-term BM suppression in a p16Ink4a/Arf-independent manner. Disclosures: No relevant conflicts of interest to declare.

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The Mitochondrial Metabolic Checkpoint and Reversing Stem Cell Aging

Blood ◽

10.1182/blood.v128.22.sci-34.sci-34 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-34-SCI-34

Author(s):

Danica Chen

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

Stem Cell ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Mitochondrial Protein ◽

Cell Aging ◽

Quiescent State ◽

Mitochondrial Oxidative Stress ◽

Hematopoietic Stem

Abstract Cell cycle checkpoints are surveillance mechanisms in eukaryotic cells that monitor the condition of the cell, repair cellular damages, and allow the cell to progress through the various phases of the cell cycle when conditions become favorable. Recent advances in hematopoietic stem cell (HSC) biology highlight a mitochondrial metabolic checkpoint that is essential for HSCs to return to the quiescent state. As quiescent HSCs enter the cell cycle, mitochondrial biogenesis is induced, which is associated with increased mitochondrial protein folding stress and mitochondrial oxidative stress. Mitochondrial unfolded protein response and mitochondrial oxidative stress response are activated to alleviate stresses and allow HSCs to exit the cell cycle and return to quiescence. Other mitochondrial maintenance mechanisms include mitophagy and asymmetric segregation of aged mitochondria. Because loss of HSC quiescence results in the depletion of the HSC pool and compromised tissue regeneration, deciphering the molecular mechanisms that regulate the mitochondrial metabolic checkpoint in HSCs will increase our understanding of hematopoiesis and how it becomes dysregulated under pathological conditions and during aging. More broadly, this knowledge is instrumental for understanding the maintenance of cells that convert between quiescence and proliferation to support their physiological functions. Disclosures No relevant conflicts of interest to declare.

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EGFR Signaling in Osteoblasts Regulates Circadian Rhythm of HSPC in Circulation

Blood ◽

10.1182/blood.v126.23.665.665 ◽

2015 ◽

Vol 126 (23) ◽

pp. 665-665

Author(s):

Sachin Kumar ◽

Jeff Vassallo ◽

Kalpana Nattamai ◽

Jose A Cancelas ◽

Hartmut Geiger

Keyword(s):

Stem Cell ◽

Circadian Rhythm ◽

Steady State ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Light Cycle ◽

Egfr Signaling ◽

Wild Type ◽

Hematopoietic Stem ◽

Organ Systems

Abstract EGFR signaling regulates growth, differentiation, proliferation and migration in multiple organ systems. We previously demonstrated that inhibition of EGFR signaling on hematopoietic stem and progenitor cells (HSPCs) enhances G-CSF induced stem cell mobilization, while preliminary data suggested that inhibition of EGFR signaling in the stem cell niche actually had the opposite effect of inhibiting mobilization (Ryan et al., Nat Med, 2010). We thus tested the novel hypothesis that there is a role for EGFR signaling in the bone marrow (BM) niche with respect to regulating hematopoiesis. We utilized a set of mouse strains that express the recombinase Cre under distinct promoters to specifically delete EGFR in various types of stroma cells including Col-Cre; EGFRf/f (deletion in osteoprogenitor/osteoblasts (OBs)), Dermo-Cre; EGFRf/f (mesenchymal stem cells (MSCs) including chondrocytes and OB), Tie2-Cre; EGFRf/f (Endothelial cells) and Nestin-Cre; EGFRf/f (Schwann/neural cells) and compared them to no Cre-EGFRf/f mice as control wild type for EGFR. Basic parameters of steady-state hematopoiesis were not altered in mice devoid of EGFR signaling in the various types of stroma cells listed above. We further investigated HSPC mobilization in EGFRf/f mice and interestingly, Col-cre and Dermo-cre EGFRf/f mice exhibited a lower number of circulating HSPC in blood in comparison to wild type mice, as determined by colony forming units (CFUs) or flow cytometry. Deletion of the EGFR in endothelial (Tie2-Cre) and neuronal (Nestin-Cre) compartments did not result in a decline in the number of circulating HSPCs. Upon G-CSF challenge, Col-cre and Dermo-cre EGFRf/f mice mobilized HSPCs similar to controls, suggesting that EGFR signaling in OBs/MSCs is dispensable for G-CSF induced mobilization. HSPCs circulation under steady state follows a circadian rhythm. We next tested whether EGFR signaling in OBs might play a role in circadian rhythm driven HSPC circulation. After 5 hours of light cycle i.e. Zeitgeber time-5 (ZT-5), when the number of circulating HSPCs is high, Col-Cre EGFRf/f and Dermo-Cre EGFRf/f mice presented with low numbers of circulating HSPCs. After 13 hours of light cycle (ZT-13) (low number of circulating HSPCs), the number of HSPCs in blood in Col-Cre EGFRf/f and Dermo-Cre EGFRf/f mice were similar to controls. Together, this suggests that EGFR signaling in OBs is essential for the rhythmic increase in circulating HSPC in blood at ZT5. Currently, we are investigating molecular mechanisms driven by EGFR signaling in OBs that regulate HSPC retention in BM and circadian regulation of EGFR signaling. In summary, our data suggest an important and also very specific (no other phenotype altered) role of EGFR signaling for regulating the circadian rhythm of HSPC circulation in peripheral blood. Disclosures No relevant conflicts of interest to declare.

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In-Depth Quantitative Multiplex Proteomics Reveal Subtype-Specific Differences Among Functionally Validated AML Stem Cell Populations

Blood ◽

10.1182/blood.v124.21.2144.2144 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2144-2144

Author(s):

Simon Raffel ◽

Jenny Hansson ◽

Carl Herrmann ◽

Christoph Lutz ◽

Eike C. Buss ◽

...

Keyword(s):

Mass Spectrometry ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Conflicts Of Interest ◽

Hierarchical Organization ◽

Differentially Expressed ◽

Cell Populations ◽

Differentially Expressed Proteins ◽

Hematopoietic Stem

Abstract Acute Myeloid Leukemia (AML) is a heterogeneous group of myeloid malignancies. The classification of AML subtypes is based on recurrent genetic abnormalities and typical histopathological features, which impact on the patient’s prognosis. AML is also the model disease for the cancer stem cell model with leukemia stem cells (LSCs) residing at the top of a hierarchical organization. LSCs have self-renewal activity and generate leukemic progeny, which make up the majority of leukemic cells. Because LSCs can be quiescent and reside in specific niches in the bone marrow, rendering them resistant to conventional chemotherapy approaches, they are considered the source of relapse. Hence, further strategies to eradicate LSCs are pivotal to improve patient outcomes of this dismal disease. LSCs present within cell populations can be detected by their capacity to re-initiate the leukemia after xenotransplantation into immuno-compromised mice. However, using current methods, it is neither possible to prospectively isolate pure functional LSCs nor to distinguish them reliably from normal hematopoietic stem cells (HSCs). To define functional LSCs we FACS-sorted primary patient samples of different AML subtypes according to surface-expression of CD34 and CD38 and transplanted each of the resulting four cell populations into conditioned NSG recipients. Several AML samples showed human leukemic engraftment in at least one of the subsets, dissecting LSC-containing and LSC-free subpopulations within the same patient. AML engraftment was mainly observed within the CD34+CD38- fraction, but several cases showed LSC activity also in the CD34+CD38+ fraction or even in the CD34- subsets. As age-matched healthy controls, we collected bone marrow hematopoietic stem and progenitor cells (HSPCs, Lineage -CD34+CD38-) from individuals older than 60 years, undergoing hip replacement surgery. In-depth quantitative multiplex proteomic analysis was performed by employing tandem mass tag (TMT) labeling and high-resolution mass spectrometry. Using this approach, more than 7,000 proteins were quantified from 10 LSC-containing and 8 LSC-free fractions from six individual AML samples of different subtypes. Importantly, our data include many low abundance proteins or others that tend to be difficult to detect by mass spectrometry, such as transcription factors and membrane proteins. We identified up to 1500 differentially expressed proteins between LSC-containing and LSC-free fractions. Most interestingly, differentially expressed proteins also clustered according to AML subtype, indicating subtype-specific proteome differences at the level of AML LSCs. Along the same lines, Gene Ontology and Gene Set Enrichment Analyses showed distinct (in some instances even opposing) differences between AML subtypes. For example, interferon- and integrin-signaling were enriched in LSC-fractions of FLT3-ITD, NPM1-mutated AMLs while reduced in LSC-fractions of FLT3-wt, NPM1-wt AML samples. In summary, our data indicate that also at the level of functionally validated LSC populations, subtype-specific differences in protein expression are remarkably evident. This heterogeneity should be taken into account with respect to the development of targeting strategies for LSCs aiming to improve the clinical outcome of AML patients. Disclosures No relevant conflicts of interest to declare.

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Establishing a Novel High-Throughput Conditioning-Free HSC Transplant Model in Zebrafish

Blood ◽

10.1182/blood-2019-127483 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4423-4423

Author(s):

Ellen Fraint ◽

Teresa V. Bowman

Keyword(s):

Stem Cell ◽

High Throughput ◽

Large Scale ◽

Fluorescent Protein ◽

Conflicts Of Interest ◽

High Fecundity ◽

Hematopoietic Stem ◽

Transplantation Model ◽

Hsc Transplantation ◽

Marrow Cells

Hematopoietic stem cell (HSC) transplantation is a widely used treatment for a range of malignant and non-malignant disorders in children and adults, although the risk of morbidity and mortality still remains unacceptably high. Preclinical modeling of HSC transplantation is critical to uncover the biological components that underlie poor patient outcomes. Zebrafish is a vertebrate animal model with high genetic and cellular conservation with human hematopoiesis with many advantages to discover regulators of HSC transplantation biology, such as high fecundity, short generation time, and lower cost of husbandry. Although prior zebrafish transplantation experiments have demonstrated feasibility, a high-throughput model that achieves robust, reproducible, high-level chimerism is lacking. We have developed a novel HSC transplantation model that fills that gap utilizing the bloodless runx1 W84X mutant zebrafish, which are devoid of endogenous HSCs. As a result, most embryos die 8-12 days post fertilization (dpf) due to the absence of definitive hematopoiesis. We hypothesized that the empty HSC niche and lack of a definitive immune system would prevent graft rejection, making robust HSC engraftment possible. We transplanted donor marrow cells that ubiquitously expressed green fluorescent protein (ubi:GFP) via intravascular injection into runx1 homozygous mutants and heterozygotes at 2dpf. Sham-injected and uninjected embryos served as negative controls. We transplanted an average of 100-200 recipients per experimental day. Survival was significantly improved in transplanted runx1 mutants, with 64% surviving in the transplanted cohort compared to 5% in the sham-injected controls, suggesting HSC transplantation likely supplied these fish with a functional hematopoietic compartment critical for survival. Donor-derived adult marrow chimerism was quantified by flow cytometry at 8 weeks post-transplantation. Successful engraftment was defined as >5% GFP+ myeloid cells. Over 99% of animals meeting this criterion also showed robust multi-lineage engraftment in the lymphoid and erythroid compartments. Over 70% of runx1 mutant recipients were engrafted compared to only 3% of the runx1 heterozygotes. The myeloid chimerism of engrafted mutant fish was 84% (+/-25%), while the single engrafted heterozygous control had only 21% myeloid chimerism. Transplanted fish remained robustly engrafted >6 months post-transplant. The runx1 mutants supported HSC self-renewal, as the GFP+ marrow cells from primary recipients were able to robustly engraft secondary runx1 mutant hosts. We also demonstrated that competitive transplantation in runx1 mutants can be used to measure HSC frequency, a critical feature needed to functionally and quantitatively assess HSC potential following genetic or pharmacological perturbations. These data demonstrate that the runx1 mutant zebrafish is an advantageous HSC transplantation host that allows quantification of long-term serially-repopulating bona fide HSCs. The advantages of our zebrafish transplantation model will allow the real-time visualization of stem cell trafficking and homing in a healthy, uninjured niche and the ability to perform large-scale screens to identify drugs that modify engraftment. This system will provide unprecedented insight into both the donor and host factors needed for robust HSC engraftment that will be helpful in improving human HSC graft outcomes. Disclosures No relevant conflicts of interest to declare.

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Hierarchical Organization of Osteoblast Reveals the Significant Role of CD166 in Hematopoietic Stem Cell Maintenance and Function

Blood ◽

10.1182/blood.v120.21.215.215 ◽

2012 ◽

Vol 120 (21) ◽

pp. 215-215

Author(s):

Brahmananda Reddy Chitteti ◽

Yinghua Cheng ◽

Melissa A. Kacena ◽

Edward F. Srour

Keyword(s):

Stem Cell ◽

Cell Function ◽

Conflicts Of Interest ◽

Hierarchical Organization ◽

Full Potential ◽

Hematopoietic Stem ◽

Runx2 Expression ◽

Culture Duration

Abstract Abstract 215 The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated (Chitteti et al. Blood 115(16):3239–48, 2010) that OB identified by their expression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166, represent a class of OB capable of mediating high levels of hematopoiesis enhancing activity (HEA). We also demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that CD166 expression tracks the maturational status of OB and is directly correlated with Runx2 expression and high HEA. Fractionation of 2d calvariae-derived OB with lineage markers (CD45, CD31, and Ter119), Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166- cells (CD166-) expressed high levels of OC suggesting that CD166- OB are more mature. We then used a co-culture system previously described by our group to assess the potential of different groups of OB to mediate HEA and sustain the expansion of clonogenic cells in culture. In vitro hematopoietic potential of bone marrow-derived Lineage-Sca1+ckit+ (LSK) cells co-cultured for 7 days with fresh OB or OB pre-cultured for 0, 1, 2, or 3 weeks (followed by an additional 1 week of co-culture with LSK cells) declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Percentage of cells expressing CD166 dropped from 63.5% at week 1 to 2.5% at week 4 (measured as total culture duration). During the same time period, the number of total colony forming units per culture dropped from 34,300 ± 4,000 (at week 1) to 1,800 ± 800 (at week 4) reflecting also the drop in the plating efficiency of cultured hematopoietic cells (22.9% ± 1.6% at week 1 versus 5.5% ± 0.5% at week 4). To assess the ability of OB to sustain stem cell function in vitro, we transplanted the progeny of LSK cells maintained in co-culture with OB for 7 days. Levels of chimerism 4mo post-transplantation in primary recipients of LSK progeny harvested from co-cultures containing Lin-Sca1-OPN+CD166+CD90+CD44+ OB were not significantly different from those in recipients of fresh LSK cells 71.8% ± 7.4% vs 86.1% ± 5.2%, respectively. Interestingly, when cells from primary recipients were transplanted into secondary recipients (4mo post primary transplantation), chimerism in mice receiving LSK progeny harvested from co-cultures containing Lin-Sca1-OPN+CD166+CD90+CD44+ OB increased to 95.8% ± 1.2% suggesting that these cells maintained the full potential of their self-renewal capacity. Chimerism levels in secondary recipients of LSK progeny harvested from co-cultures containing other fractions of OB were significantly lower than those observed for cells co-cultured with Lin-Sca1-OPN+CD166+CD90+CD44+ OB. These data suggest that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. Furthermore, these studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined phenotypic definition of OB that can mediate HEA and maintain stem cell function. Disclosures: No relevant conflicts of interest to declare.

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