scholarly journals The CD11a and EPCR marker combination simplifies and improves the purification of mouse hematopoietic stem cells

2017 ◽  
Author(s):  
Alborz Karimzadeh ◽  
Vanessa Scarfone ◽  
Connie Chao ◽  
Karin Grathwohl ◽  
John W. Fathman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gold Standard ◽  
Cell Types ◽  
The Self ◽  
Mouse Bone Marrow ◽  
Surface Markers ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Marker Combination

AbstractHematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish it from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a, and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, EPCR, can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist’s toolkit improves the purity of and simplifies isolation of HSCs.Significance StatementThe study of hematopoietic stem cells (HSCs) and their purification for transplantation requires a panel of surface markers that can be used to distinguish HSCs from other cell types. The number of markers necessary to identify HSCs continues to grow, making it increasingly difficult to identify HSCs by flow cytometry. In this study, we identified a combination of two surface markers, CD11a and EPCR, to enrich for HSCs in the mouse bone marrow without the need for additional markers. This simplified panel could aid HSC research by reducing the number of markers necessary to identify and isolate HSCs.

scholarly journals Distinct Transcriptional Signatures Distinguish the Emergence of Multipotent Progenitors and Hematopoietic Stem Cells from Endothelial Precursors in the Murine Embryo

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-8
Author(s):  
Tessa Dignum ◽  
Barbara Varnum-Finney ◽  
Sanjay Srivatsan ◽  
Stacey Dozono ◽  
Olivia Waltner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Surface Markers ◽  
Publicly Traded ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Endothelial Precursors

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSCs), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. HSCs have significant potential for use in cellular therapies and disease modeling. However, efforts to generate HSCs in vitro from pluripotent stem cells (PSCs) have been limited by an incomplete understanding of the unique phenotypic markers and transcriptional programs that distinguish HE with HSC potential. Previous studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSCs originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from HE with common phenotypic and transcriptional properties. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We previously found that the co-expression of two markers, CD61 and EPCR, identifies a subset of VE-Cadherin+ endothelial cells from the mouse P-Sp/AGM region (para-aortic splanchnopleura/aorta-gonad-mesonephros, where the first HSCs are generated from HE between E9 and E11 in development) enriched phenotypically for arterial endothelial surface markers (e.g. Dll4, CD44) and functionally for hemogenic precursors with HSC potential. However, this population remains heterogeneous, containing clonal hemogenic precursors with the potential for HSC as well as multilineage progenitor-restricted fates. Here, we report that expression of arterial marker CXCR4 further enriched for functional HSC potential in hemogenic precursors in the P-Sp/AGM between E9 and E10, when the first clonal HSC precursors are detected at rare frequency. In contrast, we detected more abundant clonal HE with multilineage hematopoietic potential (producing lymphoid, erythroid, and myeloid progeny in vitro but lacking HSC potential) at the same stage, which are distinguished by comparatively lower CXCR4 expression. To investigate transcriptional differences between HE populations differentially expressing CXCR4, we performed single-cell RNA sequencing of E9 P-Sp-derived VE-Cadherin+CD61+EPCR+ cells. Using an unbiased gene module analysis based on graph autocorrelation in the Monocle 3 platform to identify genes that co-vary over pseudotime, we found that Cxcr4 is uniquely expressed in a subset of cells simultaneously enriched for arterial-specific genes (including Dll4, Efnb2, Hey2, Sox17, Cd44) and genes with established roles in HSC maintenance and self-renewal (including Mecom, Cdkn1c, H19, Txnip, Kmt2a). Conversely, expression of these genes is decreased in cells undergoing the endothelial to hematopoietic transition at this stage based on pseudotemporal ordering, concomitant with increasing expression of hematopoietic-specifying transcription factors Runx1 and Gfi1, and other genes associated with definitive hematopoiesis (egs. Myb, Kit, Hlf, Gata2, Mpl, Lyl1). We also examined the aggregate expression of established HSC-specific signature genes from published data sets across pseudotime, and found that they exhibit similar expression dynamics to that of Cxcr4 and Dll4, reaching peak expression prior to the initiation of Runx1 and Gfi1 expression. Altogether, our studies support a model in which the initial populations of multipotent progenitors and HSCs emerge independently from HE in the P-Sp/AGM. Furthermore, our findings suggest that HE with HSC competence is uniquely defined by co-expression of arterial endothelial genes and genes encoding HSC self-renewal factors, providing insight into the earliest transcriptional programs that must be coordinated to drive HSC fate from endothelial precursors. Future studies will focus on identifying the signal pathways whose integration promotes expression of these HSC-defining transcriptional programs in endothelial cells, with the goal of advancing methods for HSC generation in vitro. Disclosures Bernstein: Lyell Immunopharma: Current equity holder in publicly-traded company, Other: Laboratory Support; Deverra Therapeutics: Current equity holder in publicly-traded company.

3065 – ETV6-RUNX1 ENHANCES THE SELF-RENEWAL OF HEMATOPOIETIC STEM CELLS AND CORRUPTS EARLY LYMPHOID DIFFERENTIATION

2020 ◽  
Vol 88 ◽  
pp. S58
Author(s):  
Mohamed Eldeeb ◽  
Jonas Ungerbäck ◽  
Mikael Sigvardsson ◽  
David Bryder
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem

scholarly journals Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Maria Rosa Lidonnici ◽  
Giulia Chianella ◽  
Francesca Tiboni ◽  
Matteo Barcella ◽  
Ivan Merelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Gene Set Enrichment Analysis ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mouse acute leukemia develops independent of self-renewal and differentiation potentials in hematopoietic stem and progenitor cells

2019 ◽  
Vol 3 (3) ◽  
pp. 419-431 ◽  
Author(s):  
Fang Dong ◽  
Haitao Bai ◽  
Xiaofang Wang ◽  
Shanshan Zhang ◽  
Zhao Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Leukemia ◽  
Progenitor Cells ◽  
Lymphoblastic Leukemia ◽  
The Self ◽  
Myelogenous Leukemia ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract The cell of origin, defined as the normal cell in which the transformation event first occurs, is poorly identified in leukemia, despite its importance in understanding of leukemogenesis and improving leukemia therapy. Although hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were used for leukemia models, whether their self-renewal and differentiation potentials influence the initiation and development of leukemia is largely unknown. In this study, the self-renewal and differentiation potentials in 2 distinct types of HSCs (HSC1 [CD150+CD41−CD34−Lineage−Sca-1+c-Kit+ cells] and HSC2 [CD150−CD41−CD34−Lineage−Sca-1+c-Kit+ cells]) and 3 distinct types of HPCs (HPC1 [CD150+CD41+CD34−Lineage−Sca-1+c-Kit+ cells], HPC2 [CD150+CD41+CD34+Lineage−Sca-1+c-Kit+ cells], and HPC3 [CD150−CD41−CD34+Lineage−Sca-1+c-Kit+ cells]) were isolated from adult mouse bone marrow, and examined by competitive repopulation assay. Then, cells from each population were retrovirally transduced to initiate MLL-AF9 acute myelogenous leukemia (AML) and the intracellular domain of NOTCH-1 T-cell acute lymphoblastic leukemia (T-ALL). AML and T-ALL similarly developed from all HSC and HPC populations, suggesting multiple cellular origins of leukemia. New leukemic stem cells (LSCs) were also identified in these AML and T-ALL models. Notably, switching between immunophenotypical immature and mature LSCs was observed, suggesting that heterogeneous LSCs play a role in the expansion and maintenance of leukemia. Based on this mouse model study, we propose that acute leukemia arises from multiple cells of origin independent of the self-renewal and differentiation potentials in hematopoietic stem and progenitor cells and is amplified by LSC switchover.

scholarly journals Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Marker Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

scholarly journals Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3758-3779 ◽  
Author(s):  
N Uchida ◽  
HL Aguila ◽  
WH Fleming ◽  
L Jerabek ◽  
IL Weissman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Critical Role ◽  
White Blood Cells ◽  
Cell Types ◽  
Dose Dependence ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery

Abstract Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca- 1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

Distinct roles of Rheb and Raptor in activating mTOR complex 1 for the self-renewal of hematopoietic stem cells

2018 ◽  
Vol 495 (1) ◽  
pp. 1129-1135 ◽  
Author(s):  
Hui Peng ◽  
Atsuo Kasada ◽  
Masaya Ueno ◽  
Takayuki Hoshii ◽  
Yuko Tadokoro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Mtor Complex 1

Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow

2006 ◽  
Vol 34 (9) ◽  
pp. 1248-1260 ◽  
Author(s):  
Oleg Tsinkalovsky ◽  
Elisabeth Filipski ◽  
Benedikte Rosenlund ◽  
Robert B. Sothern ◽  
Hans Geir Eiken ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Clock Genes ◽  
Mouse Bone Marrow ◽  
Developmentally Regulated ◽  
Hematopoietic Stem ◽  
Circadian Expression

Mac-1 and F4/80 Surface Markers Are Present on Murine Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1677-1677
Author(s):  
Toska J. Zomorodian ◽  
Debbie Greer ◽  
Kyle Wood ◽  
Bethany Foster ◽  
Delia Demers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Muscle Fibers ◽  
Stem Cell Marker ◽  
Stem Cell Markers ◽  
Surface Markers ◽  
Hematopoietic Stem ◽  
Cell Markers

Abstract Transplanted bone marrow donor cells with tissue specific phenotypes have been found in the brain, liver, heart, skin, lung, kidney, and gut of transplanted humans and mice. Such observations have led to the controversial hypothesis that hematopoietic stem cells (HSC) might be intrinsically plastic, and through transdifferentiation or fusion lead to the repair of damaged tissues throughout the body. Alternately, it is suggested that fusion of macrophages to the recipient cells may explain this phenomenon. We have shown recently that purified HSC are the cells responsible for GFP positive donor-derived muscle fibers in the recipient mice post bone marrow transplantation. However, further studies sorting for macrophage markers Mac-1 and F4/80 also resulted in donor-derived muscle fibers in the host. To address this discrepancy, we investigated subpopulations of Mac-1 and F4/80 positive cells, in the presence or absence of stem cell markers (Sca-1 and C-kit). We demonstrate that only the subpopulations of Mac-1 and F4/80 positive cells harboring stem cell markers, Sca-1 or c-kit, were capable of contributing to the regenerating muscle post transplantation. Furthermore, these same subpopulations demonstrated single cell High Proliferative Potential (HPP) (6–26%) in a 7 factor cytokine cocktail, compared to the Mac-1 or F4/80 cells with no stem cell markers (0%). Additionally, they demonstrated long-term engraftment in all three lineages at 1-year (average chimerism of 55% versus 0% in stem cell marker negative groups). These subpopulations were also evaluated for morphology using Hematoxylin/Eosin (H/E), Wright-Giemsa, and Nonspecific Esterase staining. In the Mac-1 and F4/80 positive groups, those negative for stem cell markers resembled differentiated cells of the myeloid origin (macrophages, granulocytes), while those with positive stem cell markers demonstrated stem cell characteristics. We did not observe any engraftability, donor-derived muscle fibers, or HPP potential for CD14 or cfms positive cells coexpressing stem cell markers, indicating that these markers are more appropriate for identifying macrophages. In conclusion, our studies demonstrate that both Mac-1 and F4/80 surface markers are present on HSC and therefore caution must be taken in the interpretation of data using these macrophage markers. It is reasonable to believe that the use of Mac-1 and/or F4/80 surface markers in a lineage depletion process may result in the loss of a subpopulation of stem cells, and other markers such as CD14 or c-fms may be more appropriate for eliminating differentiated macrophages.

Evidence for a Novel Blood Lineage Commitment Pathway from Lympho-Myeloid Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 802-802 ◽  
Author(s):  
Sten Eirik W. Jacobsen ◽  
Robert Mansson ◽  
Anne Hultquist ◽  
Mikael Sigvardsson ◽  
Natalija Buza-Vidas ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Lineage Commitment ◽  
Mouse Bone Marrow ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Stage Of Development ◽  
Number Of Genes

Abstract We recently identified a novel Lin−Sca-1+c-kithiCD34+Flt3hi (LSKCD34+Flt3hi) lymphoid-primed multipotent progenitor (LMPP) in adult mouse bone marrow which, although possessing a combined lymphoid (B and T cell) and myeloid (granulocyte-monocyte; GM) differentiation potential, have little or no ability to adopt erythroid (E) and megakaryocyte (MK) lineage fates (Adolfsson et al, Cell121:295, 2005). The identification of this lineage restricted lymphomyeloid progenitor implicates the existence of alternative roadmaps for lineage commitment of pluripotent hematopoietic stem cells (HSCs), distinct from the classical model suggesting that the first HSC commitment step results in a strict separation into common lymphoid and myeloid progenitors. Herein we provide further, genetic evidence for such a model. Affymetrix global gene profiling, quantitative PCR, and multiplex single cell PCR analysis of LSKCD34−Flt3− long-term (LT)-HSCs, LSKCD34+Flt3− short-term (ST)-HSCs and LSKCD34+Flt3hi LMPPs, demonstrate that LMPPs in contrast to LT-HSCs and ST-HSCs down-regulate or turn off a number of genes critically involved in MkE lineage development, including GATA-1 and the receptors for erythropoietin and thrombopoietin. In contrast, a number of genes specific for early lymphoid development, including Rag-1, sterile Ig and IL-7 receptor are upregulated in LMPPs but absent in LT-HSCs and ST-HSCs. Importantly, within the LMPP, these lymphoid genes are typically co-expressed with a number of GM associated genes such as G-CSF receptor and MPO, but virtually never co-expressed with MkE associated genes. Investigating fetal liver day 14.5 we also provide evidence for existence of the LSKCD34+Flt3hi LMPPs at this early stage of development, and using a single cell clonal assay promoting combined B, T and myeloid lineage development, we demonstrate that a large fraction of fetal LMPPs lacking MkE potential possess a combined GM, B and T cell potential. Thus, evaluation at the single cell level of combined lineage potentials and multilineage gene expression provide compelling evidence for lymphoid-priming within the HSC compartment being preceeded by a loss of MkE potential, but occurring prior to loss of GM potential.

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