Microvesicle Mediated Genetic Phenotype Modulation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4509-4509
Author(s):  
Jason M Aliotta ◽  
Mandy Pereira ◽  
Mark Dooner ◽  
Gerri Dooner ◽  
Bharat Ramratnam ◽  
...  
Keyword(s):  
Information Transfer ◽  
Conflicts Of Interest ◽  
Marrow Cell ◽  
Underlying Mechanism ◽  
Cell Phenotype ◽  
Target Cells ◽  
Specific Mrnas ◽  
Underlying Mechanisms ◽  
Specific Mrna ◽  
Marrow Cells

Abstract Abstract 4509 Objective We have previously reported that lung-derived microvesicles (MVs) can enter target marrow cells, resulting in increased levels of lung-specific mRNAs (Stem Cells 25:2245, 2007). Marrow cells which have been exposed to MVs also show increased production of pulmonary epithelial cells after transplantation into irradiated mice. The present studies have addressed the universality of the mRNA modulation and the underlying mechanisms. Methods/Results Co-culture of heart, brain, liver, and lung tissue across from murine marrow, but separated by a 0.4 micron cell-impermeable membrane, show tissue specific elevations of mRNA. MVs were found to contain lung-specific mRNA and 200 microRNAs. Proteomic studies of MVs showed up to 75 individual proteins, some of which are known to be associated with MV biogenesis and trafficking. Studies using rat/mouse hybrid cultures demonstrated that the target cell induced lung-specific mRNA elevations were mediated by transcriptional mechanisms. In these experiments, rat lung was co-cultured across from murine marrow cells and RT-PCR was performed using rat or mouse-specific primers for surfactant B. High levels of rat-specific surfactant B were seen in the co-cultured marrow cells indicating that transcription had been induced in the target cells. These conclusions were supported by additional studies employing the transcription factor inhibitors actinomycin-D and alpha-amantin. RNase treatment of conditioned media prior to marrow cell co-culture suggested that transfer of RNA may be involved in these mRNA elevations. However, our transcriptional studies indicate that we are not observing a simple transfer of MV lung-specific mRNA. One possible mechanism may be transfer of microRNA with epigenetic changes resulting in lung-specific mRNA production. Conclusion In summary, these observations suggest the existence of unique pathways for information transfer and cell phenotype determination. MV transfer could represent an underlying mechanism for much of the previous reported stem cell plasticity in different tissues. Disclosures: No relevant conflicts of interest to declare.

Cell Fate Modulation by Microvesicles: Transcriptionally-Mediated and Long Term in Nature

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4801-4801
Author(s):  
Peter J Quesenberry
Keyword(s):  
Epithelial Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Cell Phenotype ◽  
Pulmonary Epithelial Cell ◽  
Albumin Mrna ◽  
Specific Mrna ◽  
Marrow Cells

Abstract Abstract 4801 Cell-derived membrane enclosed vesicles containing mRNA, protein, microRNA, and DNA, can enter cells and effect a phenotype change. We have shown that lung-derived microvesicles enter marrow cells inducing them to express pulmonary epithelial cell-specific protein and mRNA, a variety of microRNA and to enhance their capacity to engraft in irradiated mice and express the phenotype of type II pneumocytes (Aliotta et al, Exp Hematol 38,2010). In the present studies using rat/mouse hybrid cultures and measuring species-specific mRNA, we have shown that immediately after co-culture of rat lung across from mouse marrow, mouse marrow cells express both rat and mouse specific surfactants B and C mRNA. However, when these cells are cultured in steel factor supported long-term culture, rat-specific mRNA disappears rapidly, while mouse-specific mRNA persists out to 12 weeks in liquid culture. Identical studies with rat liver cultured across from mouse marrow have shown early expression in mouse marrow of both rat and mouse albumin mRNA, but in long term in vitro culture, expression of albumin mRNA was mouse-specific. Thus, the major long-term persistent event is an alteration of transcription in the target marrow cells. In a similar fashion, marrow modulated by lung microvesicles in vitro and engrafted into lethally irradiated (950 cGy split dose) mice, evidences expression of pulmonary epithelial cell-specific mRNA or protein (surfactants) in host lung, marrow, thymus, spleen and liver 6 weeks after engraftment – the furthest time tested. These results indicate that microvesicle cell fate modulation is biologically meaningful and represents an important new mechanism for cell phenotype determination. Disclosures: No relevant conflicts of interest to declare.

Microvesicle Information Transfer from Lung to Marrow Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 437-437 ◽  
Author(s):  
Jason Aliotta ◽  
Sam Faradyan ◽  
Mark Dooner ◽  
Gerri Dooner ◽  
Peter J. Quesenberry
Keyword(s):  
Information Transfer ◽  
Fluorescent Protein ◽  
Specific Protein ◽  
Conditioned Media ◽  
Lung Epithelial Cells ◽  
Clara Cell ◽  
Normal Lung ◽  
Cell Phenotype ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract A body of work in the literature has shown that after marrow transplantation into irradiated mice, there appear cells in the lung with an epithelial lung cell phenotype, but with markers of the donor marrow cells. After transplantation of green-fluorescent protein positive marrow cells into lethally irradiated mice, we have previously shown that there are from 3–5% non-hematopoietic cells bearing the donor marrow markers in the lung, most of these being cytokeratin positive (Aliotta et al., Exp Hematol., 34(2):230–41, 2006). We have utilized a cell impermeable double culture chamber to co-culture irradiated and non-irradiated lung across from marrow cells. With 2 or 7 days of co-culture, the marrow cells express the lung-specific mRNAs surfactant proteins B and C and Clara Cell specific protein at high levels. This phenomenon is most pronounced when the lung has been exposed to 500 cGy 5 days prior to initiating co-culture, although it is also seen with non-irradiated lung. Conditioned media from lung exerts the same effects and RNase treatment of the conditioned media markedly decreased this effect. Ultracentrifugation of the conditioned media pellets the converting activity, which appears to reside in microvesicles. These microvesicles were demonstrated to enter marrow stem cells and to enhance their capacity to convert to lung epithelial cells after transplantation. Most recently, we have also shown that the capacity to enter marrow cells varies both with the cell cycle status of lineage negative Sca-1+ murine marrow cells and with the source of the microvesicles, either from irradiated or normal lung. Altogether, these data indicate that microvesicle transfer from damaged tissue may be the basis of some forms of marrow plasticity, which have been previously reported.

Lung-Derived Microvesicles Induce Stable Long-Term Epigenetic Changes In Marrow Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4799-4799
Author(s):  
Jason M Aliotta ◽  
Mandy Pereira ◽  
Ashley Amaral ◽  
Mark S Dooner ◽  
Michael DelTatto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Epithelial Cell ◽  
Bone Marrow Cells ◽  
Specific Protein ◽  
Epigenetic Changes ◽  
Specific Mrnas ◽  
Pulmonary Epithelial Cell ◽  
Specific Mrna ◽  
Marrow Cells

Abstract Abstract 4799 We have previously shown that microvesicles derived from irradiated murine lung cells enter murine bone marrow cells and induce expression of the pulmonary epithelial cell-specific mRNAs Surfactants A-D (Sp-A-D), Aquaporin-5 (Aq-5) and Clara Cell Specific Protein (CCSP). The early mRNA increases are due to direct delivery of mRNA and due to the transfer of a transcription factor to marrow cells (Aliotta JM, et al, Exp Hematol, 2010). The modulated marrow cells produce pulmonary epithelial cell-specific protein and have an enhanced ability to convert into pulmonary epithelial cells after transplantation into lethally-irradiated mice. In the present studies, we evaluated the stability of pulmonary epithelial cell-specific mRNA expression in cytokine-stimulated liquid culture and after in vivo transplantation into lethally-irradiated mice. Whole bone marrow cells isolated from C57BL/6 mice were cultured for seven days opposite C57BL/6 lung fragments, but separated from them by a 0.4 micron cell-impermeable membrane. Marrow cells were then either infused into lethally-irradiated C57BL/6 mice or established in culture with DMEM supplemented with IL-3, IL-6, IL-11 and steel factor. Engrafted mice were sacrificed six weeks after transplantation and real-time RT-PCR was carried out on marrow, liver, spleen and thymus tissue. Expression of Sp-A, B and D, CCSP and Aq-5 were variably seen in all tissues and increases over control ranged from 12-fold to 58-fold. Liquid culture was analyzed every two weeks out to 12 weeks and high levels of expression of different pulmonary epithelial cell-specific mRNAs and proteins (by western blot and Immunohistochemical staining) were observed. In order to determine the mechanism for the increased expression in culture, we established co-cultures of Fischer-344 rat lung opposite C57BL/6 mouse bone marrow cells and, using species-specific primers, determined whether the increased pulmonary epithelial cell-specific mRNA expression at each time point of secondary cytokine-supported culture was rat or mouse-specific. In every instance, the pulmonary epithelial cell-specific mRNA which was detected was mouse-specific, not rat-specific, and thus the long-term expression of lung-specific mRNA in mouse marrow was due to induced transcription by the mouse marrow cells. These data indicate that lung-derived microvesicles induce long-term stable epigenetic changes in marrow cells and suggests that the genetic changes induced by tissue microvesicles in neighboring cells may be biologically important. Disclosures: No relevant conflicts of interest to declare.

Adhesion Protein Profile of Lung-Derived Microvesicles

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4803-4803
Author(s):  
Jason M Aliotta ◽  
Napoleon A Puente ◽  
Mandy Pereira ◽  
Ashley Amaral ◽  
Mark S Dooner ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Protein Profile ◽  
Cd40 Ligand ◽  
Clara Cell ◽  
Target Cells ◽  
Lung Cells ◽  
Adhesion Protein ◽  
Adhesion Proteins ◽  
Marrow Cells

Abstract Abstract 4803 We have previously shown that murine bone marrow cells co-cultured opposite murine lung cells, but separated from them by a cell-impermeable membrane, express pulmonary epithelial cell-specific mRNAs, including Surfactants A-D, Aquaporin-5 and Clara Cell Specific Protein. This effect appears to be enhanced when the lungs used in co-culture are harvested from previously-irradiated mice. We have also shown that lung cells release membrane-bound particles called microvesicles which contain lung cell-specific proteins and mRNA. Microvesicles are capable of entering marrow cells in culture and inducing lung-specific mRNA and protein expression in marrow cells that have internalized them (Aliotta JM, et al, Stem Cells, 2007). Currently, it is not known how microvesicles gain entry into marrow cells and if this process involves the binding of specific adhesion proteins present on the marrow cells and/or the surface of microvesicles. Microvesicles derived from sources other than the lung have been described as expressing various adhesion proteins which facilitate their entry into target cells. In order to determine the presence of adhesion proteins on lung-derived microvesicles, we ultracentrifuged (100,000 g) cell-free conditioned media made from the lungs of C57BL/6 mice. Pelleted material was labeled with one of several directly-conjugated antibodies to an adhesion protein. Microvesicles which were positive for the adhesion protein of interest were identified by flow cytometry and quantified relative to its isotype control. Adhesion proteins that were found to be present on lung-derived microvesicles included CD29 (β1 integrin), CD41 (α2b integrin), CD49e (α5 integrin), CD49f (α6 integrin), CD51 (αv integrin), CD54 (ICAM-1), CD61 (β3 integrin), CD62L (L-selectin), CD81 (TAPA-1), CD107a (LAMP-1), CD154 (CD40 ligand), CD184 (CXCR4) and CD309 (VEGFR2). Those not present included CD9 (p24), CD11b (Mac-1), CD18 (β2 integrin), CD31 (PECAM-1), CD49d (α4 integrin), CD62P (P-selectin) and CD162 (PSGL-1). Lung-derived microvesicles isolated from mice that had been exposed to 500 centigrey total body irradiation five days prior to sacrifice were enriched with certain populations of adhesion protein positive microvesicles compared to those isolated from non-irradiated mice. These populations included ICAM-1 (78% increase), TAPA-1 (45% increase), CD40 ligand (108% increase) and VEGFR2 (136% increase) positive microvesicles. We are attempting to determine if lung-derived microvesicle entry into marrow cells is mediated by interactions with these adhesion proteins. These data indicate that adhesion proteins are present on lung-derived microvesicles. It is possible that differences in mRNA expression seen in marrow cells exposed to microvesicles derived from irradiated and non-irradiated lung in be related to qualitative and quantitative differences in those microvesicles. Disclosures: No relevant conflicts of interest to declare.

Marrow cell genetic phenotype change induced by human lung cancer cells

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 11108-11108
Author(s):  
P. J. Quesenberry ◽  
M. Del Tatto ◽  
D. Berz ◽  
T. Miner ◽  
T. Ng ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Carcinoma ◽  
Cancer Cells ◽  
Marrow Cell ◽  
Lung Cancer Cells ◽  
Human Lung Cancer ◽  
Type I ◽  
Lung Cancers ◽  
Specific Mrna ◽  
Marrow Cells

11108 Background: Murine lung-derived microvesicles are capable of inducing lung-specific mRNA in marrow cells, when co-cultured across from these cells, but separated from them by a cell-impermeable (0.4 micron) membrane. These converted murine marrow cells showed mRNA elevations, lung-specific protein production and enhanced capacity to convert to lung epithelial cells after in vivo transplantation into irradiated mice. We examine here whether fresh tissue from lung cancer patients would have the same capacity to genetically alter co-cultured human marrow cells. Methods: Lung cancer samples were collected from 5 patients undergoing surgery. Minced tumor tissue at 50–100 mg was co-cultured in a semi-permeable culture plate insert opposite 3.0 ×106 human marrow cells. The marrow cells were harvested after 2–7 days of co-culture. Marrow cell RNA was analyzed for lung specific mRNA using real time RT-PCR. Relative levels of gene expression was expressed a fold increase compared to level in controls. Results: Lung cancers studied were adenocarcinoma, endobronchial alveolar carcinoma, bronchioloalveolar carcinoma, non-small cell carcinoma and squamous cell carcinoma. mRNAs for aquaporin 1–5, specific for type I pneumocytes and surfactant A-D, specific for type II pneumocytes, were measured. Aquaporin I was elevated in marrow cells from co culture with all lung cancers; elevations ranging from 2.15 to 56.7 fold (mean 23 fold). Similarly surfactant B mRNA was induced in marrow cells by all lung cancers with fold elevations ranging from 7.9 to 2164 (mean fold elevation 668). More variable elevations were also seen with aquaporin 3, 4, and 5, surfactant A, surfactant C, and surfactant D. Ultracentrifugation (28,000 g) of conditioned media from these cancers revealed the presence of microvesicles with diameters of 100–180 nm. Conclusions: These observations indicate that the genetic phenotype of cells in the vicinity of lung cancer cells can be altered and that these alterations might be mediated by microvesicle transfer of genetic information. No significant financial relationships to disclose.

Contrasting underlying mechanisms of different barrier coating types

TAPPI Journal ◽  
2018 ◽  
Vol 17 (01) ◽  
pp. 31-37
Author(s):  
Bryan McCulloch ◽  
John Roper ◽  
Kaitlin Rosen
Keyword(s):  
Food Packaging ◽  
Underlying Mechanism ◽  
Consumer Products ◽  
Mechanical Degradation ◽  
Design Rules ◽  
Barrier Coatings ◽  
Barrier Materials ◽  
Barrier Coating ◽  
Coating Type ◽  
Underlying Mechanisms

Barrier coatings are used in applications including food packaging, dry goods, and consumer products to prevent transport of different compounds either through or into paper and paperboard substrates. These coatings are useful in packaging to contain active ingredients, such as fragrances, or to protect contents from detrimental substances, such as oxygen, water, grease, or other chemicals of concern. They also are used to prevent visual changes or mechanical degradation that might occur if the paper becomes saturated. The performance and underlying mechanism depends on the barrier coating type and, in particular, on whether the barrier coating is designed to prevent diffusive or capillary transport. Estimates on the basis of fundamental transport phenomena and data from a broad screening of different barrier materials can be used to understand the limits of various approaches to construct barrier coatings. These estimates also can be used to create basic design rules for general classes of barrier coatings.

scholarly journals Focus on Osteosclerotic Progression in Primary Myelofibrosis

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 122
Author(s):  
Mariarita Spampinato ◽  
Cesarina Giallongo ◽  
Alessandra Romano ◽  
Lucia Longhitano ◽  
Enrico La Spina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Marrow Cell ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Hematopoietic Stem ◽  
Clonal Proliferation ◽  
Cytokines And Chemokines ◽  
Bone Damage ◽  
Underlying Mechanisms ◽  
Modulating Functions

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hematopoietic stem-cell-derived clonal proliferation, leading to bone marrow (BM) fibrosis. Hematopoiesis alterations are closely associated with modifications of the BM microenvironment, characterized by defective interactions between vascular and endosteal niches. As such, neoangiogenesis, megakaryocytes hyperplasia and extensive bone marrow fibrosis, followed by osteosclerosis and bone damage, are the most relevant consequences of PMF. Moreover, bone tissue deposition, together with progressive fibrosis, represents crucial mechanisms of disabilities in patients. Although the underlying mechanisms of bone damage observed in PMF are still unclear, the involvement of cytokines, growth factors and bone marrow microenvironment resident cells have been linked to disease progression. Herein, we focused on the role of megakaryocytes and their alterations, associated with cytokines and chemokines release, in modulating functions of most of the bone marrow cell populations and in creating a complex network where impaired signaling strongly contributes to progression and disabilities.

scholarly journals CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
In Utero ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Show Evidence ◽  
Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

Lidocaine depresses splenocyte immune functions following trauma-hemorrhage in mice

2006 ◽  
Vol 291 (5) ◽  
pp. C1049-C1055 ◽  
Author(s):  
Takashi Kawasaki ◽  
Mashkoor A. Choudhry ◽  
Martin G. Schwacha ◽  
Kirby I. Bland ◽  
Irshad H. Chaudry
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Immune Responses ◽  
Cytokine Production ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Sham Operation ◽  
Mapk Activation ◽  
Midline Laparotomy ◽  
Marrow Cells

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6–8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and ∼90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4× shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H.

scholarly journals Transcriptome Based Projection of Single Cells to Uncover Development and Heterogeneity of Abnormal Hematopoietic Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2520-2520
Author(s):  
Parashar Dhapola ◽  
Mikael Sommarin ◽  
Mohamed Eldeeb ◽  
Amol Ugale ◽  
David Bryder ◽  
...  
Keyword(s):  
Single Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Target Cells ◽  
Cell Identity ◽  
Large Patient ◽  
Low Dimensional

Single-cell transcriptomics (scRNA-Seq) has accelerated the investigation of hematopoietic differentiation. Based on scRNA-Seq data, more refined models of lineage determination in stem- and progenitor cells are now available. Despite such advances, characterizing leukemic cells using single-cell approaches remains challenging. The conventional strategies of scRNA-Seq analysis map all cells on the same low dimensional space using approaches like tSNE and UMAP. However, when used for comparing normal and leukemic cells, such methods are often inadequate as the transcriptome of the leukemic cells has systematically diverged, resulting in irrelevant separation of leukemic subpopulations from their healthy counterpart. Here, we have developed a new computational approach bundled into a tool called Nabo (nabo.readthedocs.io) that has the capacity to directly compare cells that are otherwise unalignable. First, Nabo creates a shared nearest neighbor graph of the reference population, and the heterogeneity of this population is subsequently defined by performing clustering on the graph and calculating a low dimensional representation using t-SNE or UMAP. Nabo then calculates the similarity of incoming cells from a target population to each cell in the reference graph using a modified Canberra metric. The reference cells with higher similarity to the target cells obtain higher mapping scores. The built-in classifier is used to assign each target cell a reference cluster identity. We tested Nabo's accuracy on control datasets and found that Nabo's performance in terms of accuracy and robustness of projection is comparable to state-of-art methods. Moreover, Nabo is a generalized domain adaptation algorithm and hence can perform classification of target cells that are arbitrarily dissimilar to reference cells. Nabo could identify the cell-identity of sorted CD19+ B cells, CD14+ monocytes and CD56+ by projecting these unlabeled cells onto labelled peripheral blood mononuclear cells with an average specificity higher than 0.98. The general applicability of Nabo was demonstrated by successfully integrating pancreatic cells, sequenced in three different studies using different sequencing chemistries with comparable or better accuracy than existing methods. Also, it was conclusively demonstrated that Nabo can predict the identity of human HSPC subpopulations to the same accuracy as can be achieved by established cell-surface markers. Having Nabo at hand, we aimed to uncover the heterogeneity of hematopoietic cells from different stages of AML. Nabo showed that AML cells lacked the heterogeneity of normal CD34+ cells and were devoid of cells with HSC gene signature. A large patient-to-patient variability was found where leukemic cells mapped to distinct stages of myeloid progenitors. To ask whether this variability could reflect differences in leukemia-initiating cell identity, we induced leukemia in murine granulocyte-monocyte-lymphoid progenitors (GMLPs) using an inducible model for MLL-ENL-driven AML. On projection, more than 70% of MLL-ENL-activated cells mapped to a distinct Flt3+ subpopulation present within healthy GMLPs. Statistical validity of this projection was verified using two novel null models for testing cell projections: 1) ablated node model, wherein the mapping strength of target cells are evaluated after removal of high mapping score source nodes, and 2) high entropy features model, which rules out the background noise effect. By separating Flt3+ and Flt3- cells prior to activation of the fusion gene and performing in vitro replating assays, we could demonstrate that Flt3+ GMLPs contained 3-4 fold more leukemia-initiating cells (1/1.34 cells) than Flt3- GMLPs (1/4.89 cells), indicating that leukemia-initiating cells within GMLPs express Flt3. Taken together, Nabo represents a robust cell projection strategy for relevant analysis of scRNA-Seq data that permits an interpretable inference of cross-population relationships. Nabo is designed to compare disparate cellular populations by using the heterogeneity of one population as a point of reference allowing for cell-type specification even following perturbations that have resulted in large molecular changes to the cells of interest. As such, Nabo has critical implementation for delineation of leukemia heterogeneity and identification of leukemia-initiating cell population. Disclosures No relevant conflicts of interest to declare.

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