HSC-independent definitive hematopoietic cells persist into adult life

SummaryThe stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, various types of blood cells are already produced from hemogenic endothelial cells (HECs) before the first HSCs appear at embryonic day (E)11 in the mouse embryo. This early blood cell production from HECs, called HSC-independent hematopoiesis, includes primitive and definitive erythromyeloid progenitors that transiently support fetal blood homeostasis until HSC-derived hematopoiesis is established. Lymphoid potential has traditionally been detected in the extra-embryonic yolk sac (YS) and/or embryos before HSC emergence, but the actual presence of lymphoid progenitors at this stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent multipotent progenitors (MPPs) and HSC-independent adoptive B-lymphoid progenitors persist into adult life. Furthermore, HSCs minimally contribute to the peritoneal B-1a cell pool; most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and B-lymphoid progenitors in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

Single-Cell Analysis of Human B Lymphoid and Neutrophil/Monocyte Lineage Restriction

Lifelong production of most types of mature blood cells is sustained by a small population of cells with extensive regenerative potential. However, the detailed steps that restrict multipotent or even bipotent human hematopoietic cells to any single lineage remain poorly understood. Those that segregate the human B-lymphoid and neutrophil/monocyte (NM) lineages are of particular interest as these appear to identify a stage that might control the different properties of human leukemias that display perturbed NM and/or B-cell programs. To undertake a refined analysis of this normal lineage restriction process in human cells, we first devised a culture system that permits it to be tracked clonally and efficiently (50%). Initial experiments showed this could be achieved using a combination of multiple stromal cell types and human growth factor-supplemented medium. To elucidate the intervening transitional steps we then used multiplexed flow cytometry to compare the progeny generated in this culture system over a 2-week time course from previously defined lymphoid progenitor-enriched (P-L), NM progenitor-enriched (P-NM) and less restricted P-mix cord blood (CB) subsets. The results suggested that gain of CD45RA (RA) expression and loss of CLEC12A (C) expression appeared to accompany the sequential restriction of early CD34+ progenitors first to cells with dual NM+B-lineage potential and then just to B-lineage potential. Subsequent tracking of the lineage outputs of CD34+ RA-C- cells initially produced in larger numbers from unfractionated CD34+ CB cells either in vitro or in xenografted immunodeficient mice, confirmed the CD34+ RA-C- subset to be highly enriched in cells with dual NM+B potential. In contrast, co-generated CD45RA+ (RA+C-) and RA+CLEC12A+ (RA+C+) phenotypes displayed separate B- and NM lineage-restricted activity, respectively, as indicated by their largely exclusive clonal outputs of CD19+ pre-B and CD14+/CD15+ NM precursors. In agreement with these phenotypically established separate NM and B-lineage outputs, RA+C- cells were found to contain higher levels of B-lineage-associated gene transcripts (e.g., DNTT, CD79A, and EBF1), whereas RA+C+ cells contained higher levels of the NM transcription factor mRNAs encoded by SPI1 and CEBPA. In a further optimized stroma-free liquid culture system, the RA-C- cells could be shown to produce continuously RA+C- and RA+C+ progeny after another 3-4 days, and also RA-C- progeny which are not produced from more restricted RA+C- and RA+C+ cells, suggesting that the acquired expression of RA precedes the separation of NM and B-lineage potential that is then marked by the differential activation of C expression in RA+ cells. To examine more precisely how the process of B+NM restriction to a single lineage might be related to successive cell cycles, we labeled RA-C- cells with carboxyfluorescein diacetate succinimidyl ester (CFSE) to enable the phenotypes and growth potential of the the progeny obtained after 6 days to be directly related to their prior division histories. This revealed extensive heterogeneity in the overall distribution of the initial progeny cell cycle times but with a clear segregation in the outputs of the faster and slower dividing cells. Notably, the faster dividers produced ultimately small M+B or uni-lineage clones whereas the initially slow dividers subsequently produced larger clones, 25% of which still contained CD34+ cells or N+M+B progeny. Taken together, these findings identify new hallmark phenotypic changes that identify a critical step in the lineage restriction of primitive human hematopoietic cells with dual NM+B-lineage potential and a previously unknown association of this process with a shortening of their cell cycle transit time. Disclosures No relevant conflicts of interest to declare.

Identification of the Global miR-130a Targetome Reveals a Novel Role for TBL1XR1 in Hematopoietic Stem Cell Self-Renewal and t(8;21) AML

Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSC) point to shared core stemness properties. However, discordance between mRNA and protein signatures underscores an important role for post-transcriptional regulation by miRNAs in governing this critical nexus. Here, we identified miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impaired B lymphoid differentiation and expanded long-term HSC. Integration of protein mass spectrometry and chimeric AGO2 eCLIP-seq identified TBL1XR1 as a primary miR-130a target, whose loss of function phenocopied miR-130a overexpression. Moreover, we found that miR-130a is highly expressed in t(8;21) AML where it is critical for maintaining the oncogenic molecular program mediated by AML1-ETO. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of novel genes and molecular networks underpinning stemness properties of normal and leukemic cells.

Epigenetic regulator genes direct lineage switching in MLL-AF4 leukaemia

The fusion gene MLL-AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. However, relapse can be associated with a switch from acute lymphoblastic to acute myeloid leukaemia. Here we show that these myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate in either early, multipotent progenitors or committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes indicating that the execution and maintenance of lymphoid lineage differentiation is impaired. We show that this subversion is recurrently associated with the dysregulation of repressive chromatin modifiers, notably the nucleosome remodelling and deacetylation complex, NuRD. In addition to mutations, we show differential expression or alternative splicing of NuRD members and other genes is able to reprogram the B lymphoid into a myeloid gene regulatory network. Lineage switching in MLL-AF4 leukaemia is therefore driven and maintained by defunct epigenetic regulation.

Automated Detection Model in Classification B-Lymphoblast Cell from Normal B-Lymphoid Precursors in Blood Smear Microscopic Images Based on the Majority Voting Technique

Introduction: Acute Lymphoblastic Leukemia (ALL) is a deadly white blood cell disease that affects the human bone marrow. Detection of ALL, the most common type of leukemia, has been always riddled with complexity and difficulty in its early stages. Peripheral blood examination as a common method at the beginning of the ALL diagnosis process is a time-consuming, tedious process and greatly depends on the experts' experience, keeping up with the advances in artificial intelligence in the diagnosis process. Keeping up with the growth and development of artificial intelligence algorithms a model was developed to classify B-ALL lymphoblast cells from lymphocytes. Materials and Methods: A Fast, efficient and comprehensive model based on Deep Learning (DL) was proposed by implementing eight well-known Convolutional Neural Network (CNN) models for feature extraction on all images and evaluating in classifying B-ALL lymphoblast and Normal. After evaluating their performance, four best-performing CNN models were selected to compose an ensemble classifier, by combining the model performance of each classifier. Results: Due to the close similarity of the nuclei of cancerous and normal blood B-ALL cells, the state-of-the-art CNN models alone did not achieve acceptable performance in diagnosing these two classes and their sensitivity was low. The proposed classification model Based on the majority voting technique was adopted to combine the CNN models. The sensitivity of 99.4, the specificity of 96.7, AUC of 98.3, and accuracy of 98.5 were obtained for the proposed model. Conclusion: To classify blood cancerous cells from normal cells, the proposed method can achieve high accuracy without the intervention of the operator in cell feature determination. Thus, the DL-based model can be recommended as an extraordinary tool for the analysis of blood samples in digital laboratory equipment to assist laboratory specialists.

Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages

Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.

A bioinspired and chemically defined alternative to dimethyl sulfoxide for the cryopreservation of human hematopoietic stem cells

The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34+ progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.

Monocarboxylate transporter antagonism reveals metabolic vulnerabilities of viral-driven lymphomas

Epstein–Barr virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells—part of a phenomenon known as the “Warburg effect”— and is mediated by monocarboxylate transporters (MCTs). However, the role of MCTs has yet to be studied in EBV-associated malignancies, which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1, respectively. Functionally, MCT1 was required for early B cell proliferation, and MCT4 up-regulation promoted acquired resistance to MCT1 antagonism in LCLs. However, dual MCT1/4 inhibition led to LCL growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significantly reduced oxygen consumption rates (OCRs) and NAD+/NADH ratios, contrary to previous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/4-inhibited cancer cells. Furthermore, U-13C6–glucose labeling of MCT1/4-inhibited LCLs revealed depleted glutathione pools that correlated with elevated reactive oxygen species. Finally, we found that dual MCT1/4 inhibition also sensitized LCLs to killing by the electron transport chain complex I inhibitors phenformin and metformin. These findings were extended to viral lymphomas associated with EBV and the related gammaherpesvirus KSHV, pointing at a therapeutic approach for targeting both viral lymphomas.