Leukemic and Normal Stem Cell Transcriptional Signatures Determined by Functional Assays Are Predictive of the Overall Survival of AML Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 389-389
Author(s):  
Kolja Eppert ◽  
Katsuto Takenaka ◽  
Björn Nilsson ◽  
Eric R Lechman ◽  
Vicki Ling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Overall Survival ◽  
Stem Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Functional Assays ◽  
Stem Cell Populations

Abstract Abstract 389 Normal hematopoiesis and acute myeloid leukemia (AML) are organized as hierarchies with stem cells, which possess extensive self-renewal and proliferative capacity, at the apex. Although there is definitive evidence from experimental models for the existence of leukemic stem cells (LSC) in some human leukemias, the relevance of LSC to human disease progression is still lacking. While chemotherapeutic treatment of AML patients typically results in disease remission, the majority of patients will eventually relapse and succumb to the disease, indicating that residual LSC are not eliminated by current treatment. We hypothesize that stem cell derived gene expression profiles may be more clinically relevant than those derived from examination of bulk leukemia samples. Here we show the clinical significance of novel stem cell related expression profiles derived from 25 functionally validated human leukemia stem cell populations and 6 normal hematopoietic stem cell populations. Little is currently known about the molecular regulatory networks that govern human LSC or hematopoietic stem cells (HSC). Therefore, we have carried out global mRNA gene expression profiling of FACS sorted subpopulations of cells enriched for human stem cells, progenitor cells and mature cells from 16 AML primary patient samples and 3 cord blood samples to investigate these pathways. Similar to normal hematopoietic stem cells, leukemia stem, progenitor and mature cells can be sorted using CD34 and CD38 markers. Due to the heterogeneous nature of AML, it is vital that quantitative functional assays are used to characterize the LSC and progenitor activity in each sorted fraction. In vitro cell suspension cultures and methylcellulose colony formation assays were performed to characterize progenitor and blast populations. Importantly, we applied a novel and improved in vivo SCID leukemia initiating cell assay to substantiate the presence of LSC activity in each sorted fraction of 16 AML patient samples. With this enhanced assay, LSC were detected in the expected CD34+/CD38- population. However, in the majority of AML samples, LSC were detected in at least one additional fraction, demonstrating the importance of functional validation when interpreting global gene expression profiles of sorted stem cell populations. LSC and HSC specific signatures were identified following a statistical analysis that compared fractions with stem cell activity against those without (25 LSC vs 29 non-LSC; 6 HSC vs 6 non-HSC). When applied to an independent gene expression data set from 160 cytogenetically normal AML samples, a 25 probe LSC signature was the strongest predictor of overall survival (p<0.0001, HR=2.6, 95%CI 1.8-4.0, median survival 236 vs 999 days; Figure 1a). Furthermore, the 225 probe HSC specific signature derived from normal cells also provided a strong predictor of survival (p<0.0001, HR=2.3, 95%CI 1.5-3.4, median survival 238 vs 741 days; Figure 1b). We queried the gene expression-based chemical genomic database Connectivity Map with the LSC-related gene list and found a negative correlation between the genes in the LSC profile and the expression of genes that are transcriptionally induced following treatment with common chemotherapeutic compounds such as doxorubicin, suggesting resistance to chemotherapy as one possible mechanism for the correlation of the stem cell signatures with survival. Together these data support the hypothesis that the biological determinants that underlie stemness in both normal and leukemic cells are predictors of poor outcome, and are potential targets for novel therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Transcriptional heterogeneity of stemness phenotypes in the ovarian epithelium

2020 ◽  
Author(s):  
LE. Carter ◽  
DP. Cook ◽  
CW. McCloskey ◽  
T. Dang ◽  
O. Collins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Ovarian Surface Epithelium ◽  
Surface Epithelium ◽  
Cell Populations ◽  
Context Specific ◽  
Stem Cell Populations

AbstractThe ovarian surface epithelium (OSE) is a monolayer of epithelial cells covering the surface of the ovary. During ovulation, the OSE is ruptured to allow release of the oocyte. This wound is quickly repaired, but mechanisms of this repair are poorly understood. The contribution of tissue-resident stem cells in the homeostasis of several epithelial tissues is widely accepted, such as the intestinal epithelium, airway epithelium, and skin, but their involvement in OSE maintenance is unclear. While putative stem cell populations in the OSE have been described, how they are regulated is poorly defined. We show that traits associated with stem cells (stemness) can be increased in OSE following exposure to the cytokine TGFB1, overexpression of the transcription factor Snai1, or deletion of Brca1. By assessing the gene expression profiles of these populations, we show that stemness is often linked to mesenchymal-associated gene expression and higher activation of ERK signalling, but it is not consistently dependent on their activation. Expression profiles of these populations are extremely context specific, suggesting that stemness may not correspond to a single, distinct population, but rather is a heterogenous state that can possibly emerge from diverse environmental cues. Together, these findings support that the OSE may not require distinct stem cell populations for long-term maintenance, and may achieve this through transient dedifferentiation into a stem-like state.

scholarly journals Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2422-2430 ◽  
Author(s):  
FC Zeigler ◽  
BD Bennett ◽  
CT Jordan ◽  
SD Spencer ◽  
S Baumhueter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cell ◽  
Receptor Tyrosine Kinase ◽  
Lymphoid Cells ◽  
Stem Cell Population ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor. Cell cycle analysis shows that stem cells that do not express the receptor have a greater percentage of the population in G0 when compared with the flk-2/flt-3- positive population. Development of agonist antibodies to the receptor shows a proliferative role for the receptor in stem cell populations. Stimulation with an agonist antibody gives rise to an expansion of both myeloid and lymphoid cells and this effect is enhanced by the addition of kit ligand. These studies serve to further illustrate the importance of the flk-2/flt-3 receptor in the regulation of the hematopoietic stem cell.

Comparative gene expression analysis of zebrafish and mammals identifies common regulators in hematopoietic stem cells

Blood ◽  
2010 ◽  
Vol 115 (2) ◽  
pp. e1-e9 ◽  
Author(s):  
Isao Kobayashi ◽  
Hiromasa Ono ◽  
Tadaaki Moritomo ◽  
Koichiro Kano ◽  
Teruyuki Nakanishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Side Population ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Junction ◽  
Hematopoietic Stem

Abstract Hematopoiesis in teleost fish is maintained in the kidney. We previously reported that Hoechst dye efflux activity of hematopoietic stem cells (HSCs) is highly conserved in vertebrates, and that Hoechst can be used to purify HSCs from teleost kidneys. Regulatory molecules that are strongly associated with HSC activity may also be conserved in vertebrates. In this study, we identified evolutionarily conserved molecular components in HSCs by comparing the gene expression profiles of zebrafish, murine, and human HSCs. Microarray data of zebrafish kidney side population cells (zSPs) showed that genes involved in cell junction and signal transduction tended to be up-regulated in zSPs, whereas genes involved in DNA replication tended to be down-regulated. These properties of zSPs were similar to those of mammalian HSCs. Overlapping gene expression analysis showed that 40 genes were commonly up-regulated in these 3 HSCs. Some of these genes, such as egr1, gata2, and id1, have been previously implicated in the regulation of HSCs. In situ hybridization in zebrafish kidney revealed that expression domains of egr1, gata2, and id1 overlapped with that of abcg2a, a marker for zSPs. These results suggest that the overlapping genes identified in this study are regulated in HSCs and play important roles in their functions.

Gene Expression Profiling Reveals Fundamental Differences between Leukemic Stem Cells (Lin-CD34+CD38−) and Mature Blasts (Lin−CD34+CD38+) of Acute Myeloid Leukaemia (AML) Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1377-1377
Author(s):  
Kazem Zibara ◽  
Daniel Pearce ◽  
David Taussig ◽  
Spyros Skoulakis ◽  
Simon Tomlinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Populations ◽  
Leukemic Stem Cells ◽  
Significant Differential Expression ◽  
New Genes

Abstract The identification of LSC has important implications for future research as well as for the development of novel therapies. The phenotypic description of LSC now enables their purification and should facilitate the identification of genes that are preferentially expressed in these cells compared to normal HSC. However, gene-expression profiling is usually conducted on mononuclear cells of AML patients from either peripheral blood and/or bone marrow. These samples contain a mixture of blasts cells, normal hematopoietic cells and limited number of leukemic stem cells. Thus, this results in a composite profile that obscure differences between LSC and blasts cells with low proliferative potential. The aim of this study was to compare the gene expression profile of highly purified LSC versus leukemic blasts in order to identify genes that might have important roles in driving the leukemia. For this purpose, we analyzed the gene expression profiles of highly purified LSCs (Lin−CD34+CD38−) and more mature blast cells (Lin−CD34+CD38+) isolated from 7 adult AML patients. All samples were previously tested for the ability of the Lin−CD34+CD38− cells but not the Lin−CD34+CD38+ fraction to engraft using the non-obese diabetic/severe combined immuno-deficiency (NOD-SCID) repopulation assay. Affymetrix microarrays (U133A chip), containing 22,283 genes, were used for the analysis. Comparison of Lin-CD34+CD38- cell population to the Lin−CD34+CD38+ cell fraction showed 5421 genes to be expressed in both fractions. Comparative analysis of gene-expression profiles showed statistically significant differential expression of 133 genes between the 2 cell populations. Most of the genes were downregulated in the LSC-enriched fraction, compared to the more differentiated fraction. Gene ontology was used to determine the categories of the up-regulated transcripts. These transcripts, which are selectively expressed, include a number of known genes (e.g., receptors, signalling genes, proliferation and cell cycle genes and transcription factors). These genes play important roles in differentiation, self-renewal, migration and adhesion of HSCs. Among the genes showing the highest differences in expression levels were the following: ribonucleotide reductase M2 polypeptide, thymidylate synthetase, ZW10 interactor, cathepsin G, azurocidin 1, topoisomerase II, CDC20, nucleolar and spindle associated protein 1, Rac GTPase activating protein 1, leukocyte immunoglobulin-like receptor, proliferating cell nuclear antigen, myeloperoxidase, cyclin A1 (RRM2, TYMS, ZWINT, CTSG, AZU1, TOP2A, CDC20, NUSAP1, RACGAP1, LILRB2, PCNA, MPO, CCNA1). Some transcripts detected have not been implicated in HSC functions, and others have unknown function so far. This work identifies new genes that might play a role in leukemogenesis and cancer stem cells. It also leads to a better description and understanding of the molecular phenotypes of these 2 cell populations. Hence, in addition to being a more efficient way to further understand the biology of LSC, this should also provide a more efficient way of identifying new therapeutics and diagnostic targets.

scholarly journals Mammalian Homeobox B6 Expression Can Be Correlated With Erythropoietin Production Sites and Erythropoiesis During Development, But Not With Hematopoietic or Nonhematopoietic Stem Cell Populations

Blood ◽  
1997 ◽  
Vol 89 (8) ◽  
pp. 2723-2735 ◽  
Author(s):  
Frank Zimmermann ◽  
Ivan N. Rich
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Hox Genes ◽  
Fetal Liver ◽  
Single Gene ◽  
Lymphoid Cells ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Hox Gene ◽  
Stem Cell Populations

Abstract There has been increasing interest in the involvement of mammalian homeobox (HOX) genes in hematopoietic regulation. The HOX genes are clustered in 4 chromosomes in mice and humans. In general, 5′ end HOX gene expression is predominant in hematopoietic stem cell populations, whereas 3′ end HOX gene expression are primarily found in committed progenitor cells. Furthermore, HOX genes of the A cluster are generally found in myelomonocytic cells, B cluster genes in erythropoietic cells, and C cluster genes in lymphoid cells. The results presented here concentrate on a single gene, namely HOX B6. Preliminary observations using whole mount in situ hybridization showed that both HOX B6 and erythropoietin (EPO) gene expression occurred in exactly the same areas of the 8.5-day mouse embryo. As a consequence, we studied the expression of HOX B6 and EPO gene expression from 6.5 to 19.5 days of gestation, in the neonate, and in the adult. It was found that the sequential transfer of erythropoiesis in different organs during development was followed by a similar transfer of HOX B6 and EPO gene expression. Between days 16.5 and 17.5, both HOX B6 and EPO gene expression decrease in the fetal liver, even though hepatic erythropoiesis continues to decline and is transferred to the fetal spleen. Precisely at this time point, HOX B6 and EPO gene expression are transferred to both the fetal spleen and fetal kidney. However, surprisingly, expression of both genes increases again in the fetal liver just before birth. HOX B6 is expressed in cells from in vitro erythropoietic colonies (colony-forming unit-erythroid and burst-forming unit-erythroid) and TER-119+ erythroid cells but not in hematopoietic or nonhematopoietic stem cell populations. When the latter two populations are allowed to differentiate into erythropoietic cells, HOX B6 and erythroid-relevant markers are expressed. The results indicate that HOX B6 is intimately involved in the regulation of the erythropoietic system and could be a marker for this lineage.

scholarly journals Gene expression profiles of AML derived stem cells; similarity to hematopoietic stem cells

Leukemia ◽  
2006 ◽  
Vol 20 (12) ◽  
pp. 2147-2154 ◽  
Author(s):  
H Gal ◽  
N Amariglio ◽  
L Trakhtenbrot ◽  
J Jacob-Hirsh ◽  
O Margalit ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Hematopoietic Stem

CD244 Marks Non-Functional Hematopoietic Stem Cells with a Mast Cell Signature after Induction of Endoplasmic Reticulum Stress

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2474-2474
Author(s):  
Valgardur Sigurdsson ◽  
Shuhei Koide ◽  
Visnja Radulovic ◽  
Els Mansell ◽  
Mark Van Der Garde ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Er Stress ◽  
In Vitro Culture ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Hematopoietic Stem ◽  
Stress Induction

Hematopoietic stem cells (HSCs) are capable of replenishing the entire blood system when needed and transplantation of HSCs remains as one of the most effective, curative treatments for patients with genetic diseases and hematopoietic malignancies. In vitro culture is an essential process for ex vivo expansion and modification of HSCs, however engraftment levels of cultured HSCs cannot be accurately estimated. This is mainly due to lack of reliable cell surface markers representing functionality of HSCs after culture, which also limits the resolution of molecular analyses. We have previously shown that HSCs are vulnerable to endoplasmic reticulum (ER) stress responses fueled by accumulation of unfolded / misfolded proteins (Miharada et al., Cell Rep. 2014). Importance of ER stress suppression is also evident in vivo, as proliferative FL-HSCs fail to expand upon ER stress induction when natural molecular chaperone, bile acid, is reduced (Sigurdsson et al., Cell Stem Cell. 2016). Thus, ER stress elevation severely impairs the potential of HSCs, however usual marker profile is no longer representative of their functionality. In this study we aimed to discover the key signature and novel markers that represent functional retardation of HSC under activation and stress induction. Initially we compared gene expression profiles of fresh and 14-days cultured Lineage-Sca-1+c-kit+(LSK) CD48- (CD48-LSK) cells from mouse bone marrow using microarray analysis, since CD48 has been reported to enrich functional HSCs after in vitro culture (Noda et al., Stem Cells, 2008). We discovered abnormal up-regulations of genes frequently associated with mast cells (MC) in cultured CD48-LSK cells, and identified Cd244 as one of the top upregulated genes. CD244 is a member of the slam family of genes but is considered to be redundant with other slam markers in isolating HSCs from untreated mice. Indeed, freshly isolated CD150+CD48-LSK cells are negative for CD244. However, after 14-days in vitro culture with stem cell factor (SCF) and thrombopoietin (TPO), majority of CD150+CD48-LSK cells were positive for CD244. After shorter (7-days) culture, we found that CD48-LSK cells could be subdivided to CD244+ and CD244- populations (CD244-HSC and CD244+HSC). CD244-HSCs expressed high levels of HSC-related genes such as Fgd5, Hlf, Fhl1 and thrombopoietin receptor Mpl, In contrast, CD244+HSCs expressed MC-related genes, e.g. Cpa3, Gzmb and Mcpt8. In transplantation settings, CD244+HSCs showed no engraftment while CD244-HSCs showed long-term engraftment revealing them as functional stem cells. Since our and other groups have demonstrated that induction of ER stress impairs potential of mouse and human HSCs, we asked if ER stress induction would lead to the elevation of MC signature. Using an ER stress inducing chemical, thapsigargin, we could see increased ratio of CD244+HSCs within CD48-LSK cells. Conversely, the addition of TUDCA, a bile acid known to suppress ER stress, resulted in decreased frequency of CD244+HSCs. These findings strongly indicate that ER stress could be influencing the number of non-functional HSCs. To further substantiate the connection to ER stress and MC signature we analyzed a knock out mouse model of the ER stress modulator Trib3 (Trib3-/-) that is known to show an abnormal differentiation towards mast cells. Trib3-/- HSCs expressed MC genes including Cpa3 already at the steady-state condition. The number of CD244-HSCs after 7-days culture was significantly lower than control mice, and showed poor long-term engraftment potential in transplantation settings. To further elucidate the key molecular changes that impair HSCs, we compared gene expression profiles between fresh HSCs and CD244+/CD244-HSCs after 7-days culture. Gene expression comparison between CD244+ and CD244-HSCs independently confirmed the enrichment of MC cell related genes including Granzyme B (Gzmb), known to have negative impact on HSC potential (Carnevali et al., J Exp Med. 2014). Moreover, the Rel-A pathway was significantly lower in CD244-HSCs compared to fresh HSCs, suggesting a potential implication of NF-kB signal in the first alterations in HSCs during in vitro culture. We conclude that the induction of a MC cell signature fueled by ER stress is critical for normal HSC potential, and CD244 is a novel marker predicting the functionality of activated HSCs and allowing more detailed molecular analysis of activated HSCs. Disclosures No relevant conflicts of interest to declare.

Expression Profile of Aging Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 562-562
Author(s):  
Stuart M. Chambers ◽  
Chad A. Shaw ◽  
Margaret A. Goodell
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Expression Profile ◽  
Side Population ◽  
Expression Profiles ◽  
Cell Transplant ◽  
Side Population Cells ◽  
Hematopoietic Stem

Abstract A decline in stem cell function has been suggested to contribute to vertebrate aging. Several labs have documented a reduction in transplant efficiency and skewing in lineage contribution when murine bone marrow or hematopoietic stem cells (HSC) from old donors were transplanted into young recipients. Paradoxically, evidence from several labs including ours has shown that the percentage of phenotypically defined HSC in C57Bl/6 mice increases with age (Fig. 1). Within the serum of aged animals, systemic inflammatory markers such as C reactive protein in humans and IL-1 in mice have been shown to steadily increase with age, however it is unclear if systemic inflammation plays a role in these age-related HSC phenotypes. In order to investigate what might account for these functional changes in HSC aging, we have characterized gene expression in aged HSC using Affymetrix microarrays, examining expression profiles of HSC purified from C57Bl/6 mice that are 2-, 6-, 12-, 21-months old. Using polynomial regression over the time course, we have found more than 700 genes that are 2-fold up-regulated and more than 400 genes that are down-regulated with time. We used the Gene Ontology to categorize age-regulated genes, and have identified the category of ‘inflammatory response’ to be significantly enriched in genes that are up-regulated with age. Strikingly, several NF-Kb regulated genes, previously associated in other tissues with aging and inflammation (e.g. P-selectin, Cox-2, and ICAM-1) are up-regulated in aged HSC. The expression patterns of more than 15 genes, including clusterin, serum deprivation response, and growth hormone receptor, have been validated by quantitative real-time PCR. Furthermore, expression of several surface markers, including P-selectin, a protein that plays a role in inflammation, has been validated at the protein level by flow cytometry. In order to determine if the gene expression changes observed in aged HSC is caused by intrinsic or extrinsic factors, we have transplanted HSC from 21-month-old mice into young recipients and are assessing changes in their expression profile compared to young transplanted HSC. These data demonstrate a role for inflammation in HSC-aging, and may identify genes involved in stem cell transplant efficiency, lineage specification, and the onset of organismal aging. Figure 1: Sca-1 enriched Side Population cells at the indicated ages (in months). The percentage of cells residing within the SP increases 9-fold with age. Figure 1:. Sca-1 enriched Side Population cells at the indicated ages (in months). The percentage of cells residing within the SP increases 9-fold with age.

MixL1 Expression Identifies Putative Hematopoietic Stem Cells In the Murine Allantois.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1591-1591
Author(s):  
Adam D Wolfe ◽  
Karen Downs
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Preliminary Evidence ◽  
Yolk Sac ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Poorly Differentiated ◽  
Stem Cell Populations

Abstract Abstract 1591 MixL1, a paired-type homeodomain transcription factor, is implicated in pre-hematopoietic commitment of stem cell populations. In poorly-differentiated human lymphoma and leukemia lines, MixL1 is inappropriately over-expressed (Drakos et al., Human Pathol 2007:38:500). When constitutively expressed in mice, MixL1 is sufficient to induce Acute Myeloid Leukemia (Glaser et al., PNAS 2006:103:16460). On the basis of these observations, we hypothesize that MixL1 plays an important role in gating the cellular decision to remain in a poorly differentiated and proliferative phase rather than proceeding to definitive hematopoietic stem cell (HSC) identity. Several years ago, the placenta was shown to be a major site of hematopoiesis (Gekas et al., Dev Cell 2005:8:365; Ottersbach and Dzierzak, Dev Cell 2005:8:377). The placenta is composed of the chorionic disc and the allantois, the latter of which matures into the umbilical component of the placenta. The allantois exhibits definitive hematopoietic potential (Ziegler et al., Development 2006:133:4183; Corbel et al., Dev Biol 2007:301:478), and has recently been demonstrated to contain a core of stem cells referred to as the Allantoic Core Domain, or ACD, where potential placental hematopoietic activity may originate (Downs et al., Dev Dyn 2009:238:532). Our immediate goal is to evaluate whether MixL1 is expressed in the allantois, and to establish its precise spatiotemporal whereabouts with respect to early markers of hematopoietic cells, such as Runx1 (Chen et al., Nature 2009:457:887). Using immunohistochemistry in conjunction with the LacZ/Runx1 reporter mouse (North et al., Development 1999:126:2563), we have demonstrated that MixL1 is strongly expressed in the blood islands of the yolk sac, as well as in a broad, contiguous posterior domain of the embryo that extends to include the ACD stem cell core of the allantois. This domain does not include Runx1, and MixL1 expression temporally precedes that of Runx1 in the allantois. As development proceeds, the MixL1 signal becomes most prominent in putative nascent blood cells budding off from the poorly-described blood vessel common to the allantois, yolk sac and dorsal aortae, which we have called the “Vessel of Confluence” (VOC). Here, fetal blood is shuttled into the umbilical cord to the chorion for exchange with the mother. Shortly thereafter, Runx1 begins to appear within VOC, and is co-expressed with MixL1. These findings provide preliminary evidence that MixL1 is expressed within the allantois, and within nascent blood cells derived from a specific arterial site common to the allantois, yolk sac, and fetus. Moreover, MixL1 expression appears to precede that of Runx1. Thus, MixL1 may identify one of the earliest hematopoietic precursor cell populations thus far known in mammals. Further, these data provide additional evidence that the allantois is a promising model system for the study of definitive hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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