Prospective Isolation Research Articles

Single-cell multiomics analysis of chronic myeloid leukemia links cellular heterogeneity to therapy response.

The advent of tyrosine kinase inhibitors (TKIs) as treatment of chronic myeloid leukemia (CML) is a paradigm in molecularly targeted cancer therapy. Nonetheless, TKI-insensitive leukemia stem cells (LSCs) persist in most patients even after years of treatment and are imperative for disease progression as well as recurrence during treatment-free remission (TFR). Here, we have generated high-resolution single-cell multiomics maps from CML patients at diagnosis, retrospectively stratified by BCR::ABL1IS (%) following 12 months of TKI therapy. Simultaneous measurement of global gene expression profiles together with >40 surface markers from the same cells revealed that each patient harbored a unique composition of stem and progenitor cells at diagnosis. The patients with treatment failure after 12 months of therapy had a markedly higher abundance of molecularly defined primitive cells at diagnosis compared to the optimal responders. The multiomic feature landscape enabled visualization of the primitive fraction as a mixture of molecularly distinct BCR::ABL1+ LSCs and BCR::ABL1-hematopoietic stem cells (HSCs) in variable ratio across patients, and guided their prospective isolation by a combination of CD26 and CD35 cell surface markers. We for the first time show that BCR::ABL1+ LSCs and BCR::ABL1- HSCs can be distinctly separated as CD26+CD35- and CD26-CD35+, respectively. In addition, we found the ratio of LSC/HSC to be higher in patients with prospective treatment failure compared to optimal responders, at diagnosis as well as following 3 months of TKI therapy. Collectively, this data builds a framework for understanding therapy response and adapting treatment by devising strategies to extinguish or suppress TKI-insensitive LSCs.

Single-cell multiomics analysis of chronic myeloid leukemia links cellular heterogeneity to therapy response

The advent of tyrosine kinase inhibitors (TKIs) as treatment of chronic myeloid leukemia (CML) is a paradigm in molecularly targeted cancer therapy. Nonetheless, TKI-insensitive leukemia stem cells (LSCs) persist in most patients even after years of treatment and are imperative for disease progression as well as recurrence during treatment-free remission (TFR). Here, we have generated high-resolution single-cell multiomics maps from CML patients at diagnosis, retrospectively stratified by BCR::ABL1IS (%) following 12 months of TKI therapy. Simultaneous measurement of global gene expression profiles together with >40 surface markers from the same cells revealed that each patient harbored a unique composition of stem and progenitor cells at diagnosis. The patients with treatment failure after 12 months of therapy had a markedly higher abundance of molecularly defined primitive cells at diagnosis compared to the optimal responders. The multiomic feature landscape enabled visualization of the primitive fraction as a mixture of molecularly distinct BCR::ABL1+ LSCs and BCR::ABL1-hematopoietic stem cells (HSCs) in variable ratio across patients, and guided their prospective isolation by a combination of CD26 and CD35 cell surface markers. We for the first time show that BCR::ABL1+ LSCs and BCR::ABL1- HSCs can be distinctly separated as CD26+CD35- and CD26-CD35+, respectively. In addition, we found the ratio of LSC/HSC to be higher in patients with prospective treatment failure compared to optimal responders, at diagnosis as well as following 3 months of TKI therapy. Collectively, this data builds a framework for understanding therapy response and adapting treatment by devising strategies to extinguish or suppress TKI-insensitive LSCs.

Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

Clinical determinants of blood biomarkers of atrial cardiomyopathy: results from the ISOLATION AF ablation cohort study

Abstract Background In patients with atrial fibrillation (AF), significant advances have been made in identifying biomarkers representing diverse pathophysiological pathways, such as inflammation, myocyte injury, collagen deposition, and fatty-fibrotic infiltration. Data on clinical determinants of biomarkers in patients with AF, however, are lacking. Objective Our primary aim was to explore the relationships between clinical determinants and biomarker levels in AF patients scheduled for catheter ablation. Methods A cross-sectional analysis was conducted in AF patients undergoing catheter ablation included in the prospective multicenter ISOLATION study between July 2020 and May 2022. Clinical characteristics, routine tests and blood samples were collected before ablation. Blood samples were analyzed for known and novel cardiovascular biomarkers, including Bone morphogenetic protein 10 (BMP10), Angiopoietin-2 (Ang-2), Fibroblast growth factor 23 (FGF-23), Dickkopf-related protein 3 (DKK-3), Endocan (ESM-1), Insulin-like growth factor-binding protein 7 (IGFBP-7), B-type natriuretic peptides (proBNP, NTproBNP), High sensitive Troponin-T (hsTnT)< Growth differentiation factor 15 (GDF-15), interleukin 6 (IL-6), Fatty acid binding protein 3 (FABP3), Cancer antigen-125 (Ca-125) (Table 1). For each biomarker we trained a lasso regression model to identify the most important clinical determinants out of all the 60 available determinants (including anthropometry, ECG, comorbidities). The strongest associations are summarized in figure 1. Results Blood samples of 508 patients were analyzed. The mean age was 63 ±9 years, and 31.1% were female. 354 patients (69.7%) had paroxysmal AF and 154 patients (30.3%) had persistent AF. Heart failure was reported in 77 patients (15.2%). 355 (72.3%) patients were in sinus rhythm during sample collection. The following clinical determinants of biomarker levels were frequently identified as important: rhythm during blood phlebotomy, age, heart failure, decreased kidney function and female sex (Figure 1). Most notably, rhythm at the time of sampling was strongly associated with various biomarker levels. Female sex was positively associated with BMP10 and FGF23 (atrial fibrosis), but negatively associated with hsTNT (cardiac injury). Conclusion In patients with AF scheduled for catheter ablation, we identified several clinical determinants of biomarker levels with potential implications for disease mechanisms. In women, pro-fibrotic biomarkers were frequently higher, while male patients more often showed signs of myocardial injury. Importantly, rhythm during blood draw is a very strong determinant of many biomarkers and should be taken into account in future biomarker studies.Figure 1.Table 1.

Preprocedural atrial fibrillation burden is associated with recurrence in patients undergoing atrial fibrillation ablation: findings from the ISOLATION AF Ablation Cohort Study

Abstract Background Non-invasive quantification of electrophysiological properties of the atria is increasingly used for outcome prediction in atrial fibrillation (AF) patients. Data on the benefit of the current classification of AF compared to AF burden based on single lead ECG measurements to predict AF recurrences are scarce. Objective This study sought to assess the predictive performance of low versus high AF burden compared to the conventional AF classification for predicting recurrences after AF catheter ablation. Methods A cross-sectional analysis was conducted in AF patients scheduled for catheter ablation included in the prospective multicenter ISOLATION cohort study between July 2020 and November 2022. Clinical characteristics, routine tests, blood tests and rhythm monitoring through single lead ECGs were collected prior to ablation. ECGs were recorded three times daily for four weeks, with additional measurements in case of the onset or relief of symptoms. The primary endpoint was "ablation success" defined as freedom from documented arrhythmia after the blanking period. Of 650 included patients, pre-procedural rhythm monitoring was available in 580. 261 patients had at least 14 days of ECG measurements and completed 12 months of follow-up. In these patients, we compared the recurrences after 12 months between paroxysmal and persistent AF and high versus low AF burden through survival analysis. Maximally selected rank statistics were used to determine the best cut-off value for AF burden. Results In 261 patients, the mean age was 64 ± 9 years, and 33.7% were female. Physicians classified 170 patients (65.1%) as paroxysmal AF and 91 (34.9%) as persistent AF based on medical history. The AF burden is shown in Figure 1. The optimal calculated cut-off value for AF burden was 42%. Out of 170 patients with paroxysmal AF, 17 (10%) had a burden of > 42%. Of the 91 patients with persistent AF, 35 (38%) had a burden of < 42%. After 12 months, a significant difference in recurrences was observed between patients with paroxysmal AF versus persistent AF (p= 0.015). Patients with high AF burden, had more recurrences after 12 months, than patients with a low AF burden (p= 0.006) (Figure 2). Conclusion Our preliminary results suggest that the addition of a new classification of AF based on burden might provide additional benefit in predicting AF recurrences after catheter ablation compared to the current existing classification and may improve prognostication. Completion of patient follow-up is necessary to determine the predictive value of AF burden derived from single lead ECGs for the prediction of AF recurrence.Figure 2.

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry.

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle stem cells become activated upon muscle injury. Following activation, they proliferate, and most of their progeny differentiate to generate fusion-competent muscle cells while the remaining self-renews to replenish the stem cell pool. While the identity of muscle stem cells was defined more than a decade ago, based on the co-expression of cell surface markers, myogenic progenitors were identified only recently using high-dimensional single-cell approaches. Here, we present a single-cell mass cytometry (cytometry by time of flight [CyTOF]) method to analyze stem cells and progenitor cells in acute muscle injury to resolve the cellular and molecular dynamics that unfold during muscle regeneration. This approach is based on the simultaneous detection of novel cell surface markers and key myogenic transcription factors whose dynamic expression enables the identification of activated stem cells and progenitor cell populations that represent landmarks of myogenesis. Importantly, a sorting strategy based on detecting cell surface markers CD9 and CD104 is described, enabling prospective isolation of muscle stem and progenitor cells using fluorescence-activated cell sorting (FACS) for in-depth studies of their function. Muscle progenitor cells provide a critical missing link to study the control of muscle stem cell fate, identify novel therapeutic targets for muscle diseases, and develop cell therapy applications for regenerative medicine. The approach presented here can be applied to study muscle stem and progenitor cells in vivo in response to perturbations, such as pharmacological interventions targeting specific signaling pathways. It can also be used to investigate the dynamics of muscle stem and progenitor cells in animal models of muscle diseases, advancing our understanding of stem cell diseases and accelerating the development of therapies.

Single Cell Analysis Identifies Expression of PD-L2 on Human Bone Marrow Hematopoietic Stem Cells

The identification of specific and reliable markers for human hematopoietic stem cells (HSCs) is crucial to enable their prospective isolation allowing the investigation of unique physiological HSC functions within the blood cell hierarchy. Despite significant advancements in identification of surface markers for human bone marrow HSC isolation, the enriched populations remain heterogeneous. Here we applied single cell CITE-Seq (cellular indexing of transcriptomes and epitopes) of FACS-enriched hematopoietic stem and progenitor cells (HSPCs) to decipher differentiation hierarchies and to identify markers of most immature populations. We FACS-isolated over 60.000 high-quality human bone marrow HSPCs by sorting CD34 + and CD34 +CD38 - cells from 15 different healthy donors. These were subjected to scCITE-Seq using the BD Rhapsody system with a targeted gene panel comprising 600 most relevant genes of early HSC differentiation and 50 antibodies. To validate differentially expressed surface marker genes, we performed extensive in vitro and in vivo studies of FACS-purified subpopulations of human bone marrow and mobilized peripheral blood samples (mPB) from healthy donors. Our multi-omics based single HSPC profiling indicated Programmed death ligand 2 (PD-L2 / CD273) as a surface marker expressed on the most immature HSCs within the hematopoietic compartment defined as CD34 +CD38 -CD45RA -CD90 +. Prospective FACS-based isolation allowed a deeper understanding of transcriptomic differences depending on PD-L2 positivity. A transcriptomic principal component analysis of CD273 hi and CD273 low expressing HSCs isolated pairwise from four healthy donors proved distinct clustering accompanied by discriminative molecular signatures. Stem cell genes as HOPX, MLLT3 and MPL were enriched in CD273 hi HSCs, and CDK6 was downregulated, further validating our single-cell analysis. The differential expression of stem cell genes was validated at protein level. Single cell tracking using video-microscopy confirmed a delayed entry into cell cycle in CD273 hi HSCs, suggesting deeper quiescence of these cells. Furthermore, their mitochondrial potential was reduced. In vivo transplantations of both populations in immunocompromised NSGs are ongoing and will reveal the long-term repopulation potential of CD273 hi HSCs. Multiple in vitro differentiation assays demonstrated CD273 positivity to be associated with an increased stem cell phenotype with higher expression of EPCR (CD201) and CD90 in liquid culture, a delayed differentiation and high capability of generating multipotent colonies. Interestingly, CD273 surface expression was strongly upregulated in HSCs upon in vitro culture. Since PD-L2 is known as a ligand for the Programmed death receptor 1 (PD1), we investigated the immunomodulating function of PD-L2 expressing HSPCs by establishing an allogeneic lymphocyte reaction assay. In addition to their stem cell-like characteristics, PD-L2 expressing HSPCs mediated diminished T cell proliferation in allogeneic co-culture settings. Furthermore, cytokine profiling of co-culture supernatants indicated increased secretion of anti-inflammatory cytokines (IL-10 and TGF-β1), accompanied by reduced secretion of pro-inflammatory cytokines (IL-17A) and a diminished expression of the pro-inflammatory receptor CD69 on CD8 + T cells. These findings showed that CD34 + cells isolated from mobilized peripheral blood of healthy donors possess immunomodulating capabilities, and that PD-L2 expression indicates an immunocompromising HSC phenotype. In conclusion, this study demonstrates differential surface expression of PD-L2 within the most immature human HSCs. PD-L2 expression is associated with HSC quiescence and delayed in vitro differentiation. Furthermore, these results provide valuable insights into the immunomodulating features of HSCs expressing PD-L2.

Biomarker BMP10 profiles suggest an association of female and age with atrial endomysial fibrosis: Results from the ISOLATION AF ablation registry

Abstract Background/Introduction Atrial fibrillation has a complex pathophysiology such as inflammation, myocyte injury, collagen and lipid infiltration. Different biomarkers reflect these pathophysiological pathways. The biomarker Bone Morphogenetic Protein 10 (BMP10) is a secreted protein, expressed by myocytes, and is required for myocyte growth and development. BMP10 is associated with PITX2 and endomysial fibrosis. We recently demonstrated that BMP10 is independently associated with left atrial andomysial fibrosis. Purpose To analyze the correlation between the blood-based biomarker BMP10 with sex and age in patients with atrial fibrillation. Methods A cross-sectional analysis was conducted of atrial fibrillation patients scheduled for catheter ablation included from July 2020 through May 2022 in the prospective multicenter ISOLATION cohort study (NCT04342312). Clinical characteristics and routine tests were collected before ablation. Blood samples were taken before ablation and were analyzed for BMP10 among other known and novel cardiovascular biomarkers. We investigated the correlation between BMP10 and age in a univariate analysis and in a multivariate analysis including differences in clinical traits. In addition, we investigated the difference in BMP10 levels between males and females. Results Blood samples from 509 patients were analyzed. Their mean age was 64±9 years, and 31.2% were female. 352 patients (69.2%) had paroxysmal AF and 152 patients (29.9%) had persistent AF. Heart failure was reported in 77 patients (15%). 357 (70%) patients were in sinus rhythm at the time the blood was drawn (table 1). Univariate analysis showed that BMP10 levels increased with age (figure 1). After adjusting for sex, type of AF, heart failure and rhythm at blood drawing, BMP10 remained higher in older patients (Beta coefficient 0.77, standard error 0.14, p-value <0.001). In addition, univariate analysis showed that BMP10 was significantly higher in women than in men (2.16 ng/L versus 1.95 ng/l, P< 0.001). After adjusting for age, BMP10 remained higher in women than in men. Conclusion Elevated BMP10 levels suggest that in older patients possibly more atrial fibrosis is present and thus more substrate for AF. In addition, the different biomarker profile suggests that AF in women may more often be associated with atrial fibroses than in men.

Early Molecular Steps in Human Bone Marrow Stem Cell Differentiation Trajectories

Understanding the molecular signatures of human hematopoietic stem/progenitor cells (HSCPs) and gene network changes leading to differentiation induction are of utmost importance to achieve therapeutic hematopoietic stem cell (HSC) expansion. The identification of surface markers has improved the prospective isolation of human bone marrow HSCs over the last decades, but the isolated population still remains very heterogeneous. Single cell sequencing approaches have the potential to unbiased assort stem cells along their differentiation stages and trajectories, and to decipher molecular markers in differentiation hierarchies. However, whole transcriptome single cell sequencing often suffers from inadequate detection of low-expressed genes with increased drop-outs. To address this limitation and to provide sensitive mRNA quantification in human HSPCs, we developed a selected targeted gene panel (600 genes) in combination with 50 protein markers to cover most relevant genes/proteins of early HSPC differentiation and cell cycle status. We employed single cell CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) based multi-omic profiling to >60.000 high-quality FACS-enriched human bone marrow HSPCs (CD34 + and CD34 +CD38 - cells) from 15 healthy donors representing young, mid and old age groups. Here, we demonstrate the most comprehensive molecular map of early human HSC differentiation and provide evidence of early branching points into lineage trajectories across different age groups using Slingshot pseudotime analysis. Our data demonstrate that the HSC branched into two main lineages, with the first branching point giving rise to megakaryocytic and erythroid progenitors (MKP/ERP), followed by lymphoid and myeloid progenitors (LMP/MDP). As expected, the most immature HSCs were defined by high expression of CD34, CD90, CRHBP, HOPX, HLF, MLLT3. However, we observed a number of hitherto undescribed HSC markers. The MKP/ERP lineage was defined by GATA1, TFRC, CD36, MPL and VWF expression. Furthermore, we found CD274, the Programmed Cell Death receptor ligand 1, to be selectively expressed in the MKP cluster. Re-clustering and trajectory analysis of the most immature CD34 +CD38 -/low cells confirmed early lineage commitment, suggesting that an MKP/ERP development does not include myeloid or lymphoid progenitors. A similar pattern was observed for all age groups. Interestingly, we observed increased expression of novel marker genes ( HLA-E, DLK1, ADGRG6, ADGRG1, MMRN1) and proteins (CD137 and CD273) in the most immature HSCs. Besides a gradual upregulation of CD38 along differentiation, differentiated cells showed lower CD45 levels. Furthermore, MKP/ERP committed cells showed a downregulation of SPINK2. TradeSeq analysis defined the continuous single cell expression changes in genes and markers upon early differentiation and lineage commitment, allowing the detection of decision-making gene networks. Our data adds further evidence to the revised model of hematopoiesis that challenges the dogma of a common myeloid progenitor giving rise to erythroid, megakaryocytic and myeloid cells. Furthermore, we see a structured hierarchical organization with a sequential loss of lineage potential and not an early commitment of uni-lineage restricted precursors. We report on the upregulation of ADGRG6, ADGRG1, DLK1, HLA-E, MMRN1 CD137 and CD273 in the most immature HSCs. Molecular and functional characterization of CD273 hi and low HSCs are presented in an adjacent report by our group at this meeting (Schmachtel et al.). In conclusion, we provide a valuable resource for the continuous regulation of genes and surface proteins along early differentiation of very immature human HSCs.

Combination of CD49b and CD229 Reveals a Subset of Multipotent Progenitors With Short-Term Activity Within the Hematopoietic Stem Cell Compartment.

Hematopoiesis is maintained by hematopoietic stem cells (HSCs) that replenish all blood lineages throughout life. It is well-established that the HSC pool is functionally heterogeneous consisting of cells differing in longevity, self-renewal ability, cell proliferation, and lineage differentiation. Although HSCs can be identified through the Lineage-Sca-1+c-Kit+CD48-CD34-CD150+ immunophenotype, the cell surface marker combination does not permit absolute purification of functional HSCs with long-term reconstituting ability. Therefore, prospective isolation of long-term HSCs is crucial for mechanistic understanding of the biological functions of HSCs and for resolving functional heterogeneity within the HSC population. Here, we show that the combination of CD229 and CD49b cell surface markers within the phenotypic HSC compartment identifies a subset of multipotent progenitor (MPP) cells with high proliferative activity and short-term reconstituting ability. Thus, the addition of CD229 and CD49b to conventional HSC markers permits prospective isolation of functional HSCs by distinguishing MPPs in the HSC compartment.

Isolation of Tissue Macrophages in Adult Zebrafish.

Tissue macrophages are essential components of the immune system that also play key roles in vertebrate development and homeostasis, including in zebrafish, which has gained popularity over the years as a translational model for human disease. Commonly, zebrafish macrophages are identified based on expression of fluorescent transgenic reporters, allowing for real-time imaging in living animals. Several of these lines have also proven instrumental to isolate pure populations of macrophages in the developing embryo and larvae using fluorescence-activated cell sorting (FACS). However, the identification of tissue macrophages in adult fish is not as clear, and robust protocols are needed that would take into account changes in reporter specificity as well as the heterogeneity of mononuclear phagocytes as fish reach adulthood. In this chapter, we describe the methodology for analyzing macrophages in various tissues in the adult zebrafish by flow cytometry. Coupled with FACS, these protocols further allow for the prospective isolation of enriched populations of tissue-specific mononuclear phagocytes that can be used in downstream transcriptomic and/or epigenomic analyses. Overall, we aim at providing a guide for the zebrafish community based on our expertise investigating the adult mononuclear phagocyte system.

MiR-126 identifies a quiescent and chemo-resistant human B-ALL cell subset that correlates with minimal residual disease.

Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. Recent evidence has implicated subpopulations of B-ALL cells with stem-like features in disease persistence. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intratumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. We exploited patient-derived xenograft (PDX) models with B-ALL cells transduced with a miR-126 reporter allowing the prospective isolation of miR-126(high) cells for their functional and transcriptional characterization. Discrete miR-126(high) populations, often characterized by MIR126 locus demethylation, were identified in 8/9 PDX models and showed increased repopulation potential, in vivo chemotherapy resistance and hallmarks of quiescence, inflammation and stress-response pathway activation. Cells with a miR-126(high) transcriptional profile were identified as distinct disease subpopulations by single-cell RNA sequencing in diagnosis samples from adult and pediatric B-ALL. Expression of miR-126 and locus methylation were tested in several pediatric and adult B-ALL cohorts, which received standardized treatment. High microRNA-126 levels and locus demethylation at diagnosis associate with suboptimal response to induction chemotherapy (MRD > 0.05% at day +33 or MRD+ at day +78).

Tetraspanin CD82 Associates with Trafficking Vesicle in Muscle Cells and Binds to Dysferlin and Myoferlin.

Tetraspanins organize protein complexes at the cell membrane and are responsible for assembling diverse binding partners in changing cellular states. Tetraspanin CD82 is a useful cell surface marker for prospective isolation of human myogenic progenitors and its expression is decreased in Duchenne muscular dystrophy (DMD) cell lines. The function of CD82 in skeletal muscle remains elusive, partly because the binding partners of this tetraspanin in muscle cells have not been identified. CD82-associated proteins are sought to be identified in human myotubes via mass spectrometry proteomics, which identifies dysferlin and myoferlin as CD82-binding partners. In human dysferlinopathy (Limb girdle muscular dystrophy R2, LGMDR2) myogenic cell lines, expression of CD82 protein is near absent in two of four patient samples. In the cell lines where CD82 protein levels are unaffected, increased expression of the ≈72kDa mini-dysferlin product is identified using an antibody recognizing the dysferlin C-terminus. These data demonstrate that CD82 binds dysferlin/myoferlin in differentiating muscle cells and its expression can be affected by loss of dysferlin in human myogenic cells.

Reprogramming of Acute Myeloid Leukemia Patients Cells: Harboring Cancer Mutations Requires Targeting of AML Hierarchy.

Screening of primary patient acute myeloid leukemia (AML) cells is challenging based on intrinsic characteristics of human AML disease and patient-specific conditions required to sustain AML cells in culture. This is further complicated by inter- and intra-patient heterogeneity, and "contaminating" normal cells devoid of molecular AML mutations. Derivation of induced pluripotent stem cells (iPSCs) from human somatic cells has provided approaches for the development of patient-specific models of disease biology and has recently included AML. Although reprogramming patient-derived cancer cells to pluripotency allows for aspects of disease modeling, the major limitation preventing applications and deeper insights using AML-iPSCs is the rarity of success and limited subtypes of AML disease that can be captured by reprogramming to date. Here, we tested and refined methods including de novo, xenografting, naïve versus prime states and prospective isolation for reprogramming AML cells using a total of 22 AML patient samples representing the wide variety of cytogenetic abnormalities. These efforts allowed us to derive genetically matched healthy control (isogenic) lines and capture clones found originally in patients with AML. Using fluorescently activated cell sorting, we revealed that AML reprogramming is linked to the differentiation state of diseased tissue, where use of myeloid marker CD33 compared to the stem cell marker, CD34, reduces reprogramming capture of AML+ clones. Our efforts provide a platform for further optimization of AML-iPSC generation, and a unique library of iPSC derived from patients with AML for detailed cellular and molecular study.

Abstract 606: Ischemic Rescue By Transplantation Of Vascular Progenitor Cells Isolated From Fat

Background: Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia. Methods: Actin CreER /R26 VT2/GK3 “Rainbow” reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA sequencing was used to characterize distinct vessel-forming populations and their interactions. Results: Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells (MSCs): VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105 high Sca1 low , which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105 low Sca1 high and forms stunted vessels and fat. Interestingly, co-transplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice. Conclusions: These findings suggest that autologous co-transplantation of synergistic VSPCs from non-essential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

Differential expression of endothelial protein C receptor (EPCR) in hematopoietic stem and multipotent progenitor cells in young and old mice

Endothelial protein C receptor (EPCR) has emerged as one of the most conserved and reliable surface markers for the prospective identification and isolation of hematopoietic stem cells (HSCs). Prior studies have consistently demonstrated that EPCR expression enriches HSCs capable of long-term multilineage repopulation in both mouse and human across different hematopoietic tissues, including bone marrow (BM), fetal liver and ex vivo HSC expansion cultures. However, little is known about the expression profiles of EPCR in multipotent progenitor (MPP) populations located immediately downstream of HSCs in the hematopoietic hierarchy and which play a major role in sustaining lifelong blood cell production. Here, we incorporate EPCR antibody detection into a multi-parameter flow cytometric panel, which allows accurate identification of HSCs and five MPP subsets (MPP1-5) in mouse BM. Our data reveal that all MPP populations contain EPCR-expressing cells. Multipotent MPP1 and MPP5 contain higher proportion of EPCR+ cells compared to the more lineage-biased MPP2–4. Notably, high expression of EPCR enriches phenotypic HSC and MPP5, but not MPP1. Comparison of EPCR expression profiles between young and old BM reveals ageing mediated expansion of EPCR-expressing cells only in HSCs, but not in any of the MPP populations. Collectively, our study provides a comprehensive characterization of the surface expression pattern of EPCR in mouse HSC and MPP1–5 cells during normal and aged hematopoiesis.

Combination of Distinct Vascular Stem/Progenitor Cells for Neovascularization and Ischemic Rescue.

Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia. The ActinCreER/R26VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions. Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice. These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

Rearrangement of cell types in the rat carotid body neurogenic niche induced by chronic intermittent hypoxia.

The carotid body (CB) is a prototypical acute oxygen (O2 )-sensing organ that mediates reflex hyperventilation and increased cardiac output in response to hypoxaemia. CB overactivation, secondary to the repeated stimulation produced by the recurrent episodes of intermittent hypoxia, is believed to contribute to the pathogenesis of sympathetic hyperactivity present in sleep apnoea patients. Although CB functional plasticity induced by chronic intermittent hypoxia (CIH) has been demonstrated, the underlying mechanisms are not fully elucidated. Here, we show that CIH induces a small increase in CB volume and rearrangement of cell types in the CB, characterized by a mobilization of immature quiescent neuroblasts, which enter a process of differentiation into mature, O2 -sensing and neuron-like, chemoreceptor glomus cells. Prospective isolation of individual cell classes has allowed us to show that maturation of CB neuroblasts is paralleled by an upregulation in the expression of specific glomus cell genes involved in acute O2 -sensing. CIH enhances mitochondrial responsiveness to hypoxia in maturing neuroblasts as well as in glomus cells. These data provide novel perspectives on the pathogenesis of CB-mediated sympathetic overflow that may lead to the development of new pharmacological strategies of potential applicability in sleep apnoea patients. KEY POINTS: Obstructive sleep apnoea is a frequent condition in the human population that predisposes to severe cardiovascular and metabolic alterations. Activation of the carotid body, the main arterial oxygen-sensing chemoreceptor, by repeated episodes of hypoxaemia induces exacerbation of the carotid body-mediated chemoreflex and contributes to sympathetic overflow characteristic of sleep apnoea patients. In rats, chronic intermittent hypoxaemia induces fast neurogenesis in the carotid body with rapid activation of neuroblasts, which enter a process of proliferation and maturation into O2 -sensing chemoreceptor glomus cells. Maturing carotid body neuroblasts and glomus cells exposed to chronic intermittent hypoxia upregulate genes involved in acute O2 sensing and enhance mitochondrial responsiveness to hypoxia. These findings provide novel perspectives on the pathogenesis of carotid body-mediated sympathetic hyperactivation. Pharmacological modulation of carotid body fast neurogenesis could help to ameliorate the deleterious effects of chronic intermittent hypoxaemia in sleep apnoea patients.

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming "units" known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Unraveling unique features of plasma cell clones in POEMS syndrome with single-cell analysis.

POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.