Tie2Cre-mediated gene ablation defines the stem-cell leukemia gene (SCL/tal1)–dependent window during hematopoietic stem-cell development

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3871-3874 ◽  
Author(s):  
Thorsten M. Schlaeger ◽  
Hanna K. A. Mikkola ◽  
Christos Gekas ◽  
Hildur B. Helgadottir ◽  
Stuart H. Orkin
Keyword(s):  
Stem Cell ◽  
Blood Cells ◽  
Fetal Liver ◽  
Cell Leukemia ◽  
Hematopoietic Stem ◽  
Gene Ablation ◽  
Epidermal Growth ◽  
Fetal Hematopoiesis ◽  
Stem Cell Leukemia

AbstractThe stem-cell leukemia gene (SCL/tal1) is essential for the formation of all blood lineages. SCL is first expressed in mesodermal cells that give rise to embryonic blood cells, and continues to be expressed in fetal and adult hematopoietic stem cells (HSCs). However, SCL is not required for the maintenance of established long-term repopulating (LTR) HSCs in the adult. The time point at which HSC development becomes SCL independent has not been defined. Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains–2 (Tie2) expression appears in hemogenic and vasculogenic sites shortly after SCL. We therefore used the Tie2Cre mouse to inactivate SCL early during embryonic and fetal hematopoiesis. Tie2Cre completely inactivated SCL in yolk sac, the aortagonad-mesonephros (AGM) region, and fetal liver hematopoietic cells and circulating blood cells. However, the fetal liver was colonized by functional LTR-HSCs. Yet SCL remained crucial for proper differentiation of both primitive and definitive red cells and megakaryocytes. These results indicate that the SCL-dependent phase of HSC development ends before Tie2Cre-mediated gene ablation becomes effective.

Stem cell leukemia protein directs hematopoietic stem cell fate

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3336-3341 ◽  
Author(s):  
Atsushi Kunisato ◽  
Shigeru Chiba ◽  
Toshiki Saito ◽  
Keiki Kumano ◽  
Etsuko Nakagami-Yamaguchi ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Dominant Negative ◽  
Cell Leukemia ◽  
Hematopoietic Stem ◽  
Fetal Thymus ◽  
Lymphoid Lineage ◽  
Stem Cell Leukemia

Abstract Stem cell leukemia (SCL) protein has been shown to be an essential transcription factor during hematopoietic development in the embryo. In adult hematopoiesis, however, the role for SCL has remained largely unknown, whereas it is expressed in bone marrow hematopoietic stem cells (HSCs). In this study, we performed HSC transplantation and an in vitro HSC differentiation assay using retrovirally transduced HSCs with wild-type (WT) and dominant-negative (DN) SCL. The transplantation experiments showed that SCL does not affect the long-term repopulating capacity of HSCs but that WT SCL and DN SCL increase the short-term contribution of the transduced HSCs in myeloid and lymphoid lineages, respectively. An in vitro single-cell assay using a fetal thymus organ culture system further demonstrated that WT SCL facilitates HSCs to differentiate into the myeloid lineage but that DN SCL facilitates HSCs to differentiate into the lymphoid lineage. We conclude that the up-regulation or down-regulation of SCL directs HSCs toward myeloid or lymphoid lineage, respectively, although SCL does not affect their long-term repopulating capacity. (Blood. 2004;103: 3336-3341)

scholarly journals Functional but Abnormal Adult Erythropoiesis in the Absence of the Stem Cell Leukemia Gene

2005 ◽  
Vol 25 (15) ◽  
pp. 6355-6362 ◽  
Author(s):  
Mark A. Hall ◽  
Nicholas J. Slater ◽  
C. Glenn Begley ◽  
Jessica M. Salmon ◽  
Leonie J. Van Stekelenburg ◽  
...  
Keyword(s):  
Stem Cell ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Target Genes ◽  
Growth Defect ◽  
Unexpected Finding ◽  
Cell Leukemia ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Stem Cell Leukemia

ABSTRACT Previous studies have indicated that the stem cell leukemia gene (SCL) is essential for both embryonic and adult erythropoiesis. We have examined erythropoiesis in conditional SCL knockout mice for at least 6 months after loss of SCL function and report that SCL was important but not essential for the generation of mature red blood cells. Although SCL-deleted mice were mildly anemic with increased splenic erythropoiesis, they responded appropriately to endogenous erythropoietin and hemolytic stress, a measure of late erythroid progenitors. However, SCL was more important for the proliferation of early erythroid progenitors because the predominant defects in SCL-deleted erythropoiesis were loss of in vitro growth of the burst-forming erythroid unit and an in vivo growth defect revealed by transplant assays. With respect to erythroid maturation, SCL-deleted proerythroblasts could generate more mature erythroblasts and circulating red blood cells. However, SCL was required for normal expression of TER119, one of the few proposed target genes of SCL. The unexpected finding that SCL-independent erythropoiesis can proceed in the adult suggests that alternate factors can replace the essential functions of SCL and raises the possibility that similar mechanisms also explain the relatively minor defects previously observed in SCL-null hematopoietic stem cells.

Identification of a Developmentally-Restricted Hematopoietic Stem Cell That Gives Rise to Innate-like Lymphocytes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4302-4302
Author(s):  
Anna E Beaudin ◽  
Scott W. Boyer ◽  
Gloria Hernandez ◽  
Camilla E Forsberg
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Lineage Tracing ◽  
Lymphoid Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem

Abstract The generation of innate-like immune cells distinguishes fetal hematopoiesis from adult hematopoiesis, but the cellular mechanisms underlying differential cell production during development remain to be established. Specifically, whether differential lymphoid output arises as a consequence of discrete hematopoietic stem cell (HSC) populations present during development or whether the fetal/neonatal microenvironment is required for their production remains to be established. We recently established a Flk2/Flt3 lineage tracing mouse model wherein Flk2-driven expression of Cre recombinase results in the irreversible switching of a ubiquitous dual-color reporter from Tomato to GFP expression. Because the switch from Tom to GFP expression in this model involves an irreversible genetic excision of the Tomato gene, a GFP+ cell can never give rise to Tom+ progeny. Using this model, we have definitively demonstrated that all functional, adult HSC remain Tomato+ and therefore that all developmental precursors of adult HSC lack a history of Flk2 expression. In contrast, adoptive transfer experiments of Tom+ and GFP+ fetal liver Lin-cKit+Sca1+ (KLS) fractions demonstrated that both Tom+ and GFP+ fetal HSC support serial, long-term multilineage reconstitution (LTR) in irradiated adult recipients. We have therefore identified a novel, developmentally restricted HSC that supports long-term multilineage reconstitution upon transplantation into an adult recipient but does not normally persist into adulthood. Developmentally-restricted GFP+ HSC display greater lymphoid potential, and regenerated both innate-like B-1 lymphocytes and Vg3-expressing T lymphocytes to a greater extent than coexisting Tom+ FL and adult HSC. Interestingly, whereas developmental regulation of fetal-specific B-cell subsets appears to be regulated cell-instrinsically, as fetal HSC generated more innate-like B-cells than adult HSC even within an adult environment, T-cell development may be regulated both cell intrinsically and extrinsically, as both the cell-of-origin and the fetal microenvironment regulated the generation of innate-like T-cells. Our results provide direct evidence for a developmentally restricted HSC that gives rise to a layered immune system and describes a novel mechanism underlying the source of developmental hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, these findings are critical for understanding the development of autoimmune diseases, allergies, and tolerance induction upon organ transplantation. Furthermore, by uncoupling self-renewal capacity in situ with that observed upon transplantation, our data suggests that transplantation- and/or irradiation-induced cues may allow for the engraftment of developmental HSC populations that do not normally persist in situ. As LTR upon transplantation has served as the prevailing definition of adult HSC origin during development, our data challenge the current conceptual framework of adult HSC origin. Disclosures No relevant conflicts of interest to declare.

Single administration of stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination soon after irradiation prevents nonhuman primates from myelosuppression: long-term follow-up of hematopoiesis

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 878-885 ◽  
Author(s):  
Michel Drouet ◽  
Frédéric Mourcin ◽  
Nancy Grenier ◽  
Valérie Leroux ◽  
Josianne Denis ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Dose ◽  
Gamma Irradiation ◽  
Blood Cells ◽  
Stem Cell Factor ◽  
Growth And Development ◽  
Hematopoietic Stem ◽  
Interleukin 3 ◽  
Development Factor

Abstract Preservation of hematopoietic stem and progenitor cell survival is required for recovery from radiation-induced myelosuppression. We recently showed that short-term injection of antiapoptotic cytokine combinations into mice soon after lethal gamma irradiation promoted survival. The present study investigated the hematopoietic response of cynomolgus monkeys to a single dose of stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination (4F, each factor given intravenously at 50 μg/kg) administered 2 hours after 5-Gy gamma irradiation. Treated monkeys (n = 4) experienced no thrombocytopenia. Only 1 in 4 displayed a transient period of neutropenia (neutrophil [ANC] count < 0.5 × 109/L), whereas all irradiated controls (n = 4) experienced neutropenia (5-12 days) and thrombocytopenia (platelet [PLT] count < 20 × 109/L, 5-31 days). Treated animals exhibited an impressive 2-wave PLT response that peaked at days 8 and 22 after total body irradiation (TBI). Areas under the curve (AUC) of PLTs, ANCs, white blood cells (WBCs), and red blood cells (RBCs) between days 0 and 90 were significantly higher in treated animals than in controls. Humeral bone marrow–derived clonogenic activity was significantly spared at 24 hours and 4 days after TBI in treated monkeys. No apparent impairment of the hematopoietic status and stem cell pool, in terms of long-term culture–initiating cells (LTC-ICs) and side population (SP) cells, was observed after 15 months. These results strongly suggest that the 4F cytokine combination, as a single dose regimen, could act as an emergency treatment for nuclear accident or terrorism victims.

scholarly journals An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Na Yoon Paik ◽  
Grace E. Brown ◽  
Lijian Shao ◽  
Kilian Sottoriva ◽  
James Hyun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem ◽  
Main Site

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation. While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC. Disclosures No relevant conflicts of interest to declare.

Long-Term Follow-Up of Serially Transplanted Mice Receiving Extended Reduced Intensity Selection of MGMT-P140K Expressing Murine Repopulating Stem Cells Demonstrates Stable Oligo- to Polyclonal Hematopoiesis without Clonal Exhaustion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1286-1286
Author(s):  
Claudia Ball ◽  
Manfred Schmidt ◽  
Ingo Pilz ◽  
Monika Schrempp ◽  
Christof von Kalle ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Reduced Intensity ◽  
Selection Of

Abstract In vivo selection of gene modified hematopoietic stem cells permanently increases the relative proportion of blood cells that carry a therapeutic transgene despite initially low gene transfer efficiency, thereby decreasing the likelihood of insertional mutagenesis and avoiding the need of myeloablative conditioning regimens. P140K Mutant O6-methylguanine-DNA methyltransferase (MGMT) enzyme confers resistance to the combination of the MGMT inhibitor O(6)-benzylguanine (O(6)BG) and nitrosourea drugs such as 1,3-bis-(2 chloroethyl)-1-nitrosourea (BCNU). We have previously shown that reduced intensity and toxicity BCNU/O6-BG selection allows efficient selection of MGMT-P140K expressing oligoclonal murine hematopoiesis. Nevertheless, whether long-term selection and the associated proliferative stress impairs long-term differentiation and proliferation of MGMT-P140K expressing stem cell clones is currently unknown and remains a major concern in the clinical application of MGMT selection. To address this question, serial transplantations of murine MGMT-P140K expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were done. After ex vivo gene transfer of an MGMT/IRES/eGFP encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and primary, secondary and tertiary recipient mice were subsequently treated every four weeks in order to exaggerate potential effects on long-term clonal behaviour. Lineage contribution of the transduced hematopoiesis was monitored by FACS over a total of 14 rounds of selection and clonality by LAM-PCR over a total of 12 rounds of selection. In primary mice the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without persisting peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential of transduced cells in 1st, 2nd and 3rd generation animals. LAM PCR analysis of peripheral blood samples revealed stable oligo- to polyclonal hematopoiesis in primary and secondary mice. Evidence for predominant clones or clonal exhaustion was not observed despite up to 12 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice that received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis, indicating extensive self-renewal of transplantable stem cells in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K expressing hematopoietic stem cells by repetitive chemotherapy does not affect their differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies for the amplification of a limited number of gene corrected clones in clinical gene therapy.

The Endothelial Antigen ESAM Marks Hematopoietic Stem Cells throughout Life

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 727-727 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Stefan Butz ◽  
Koichi Kokame ◽  
Paul W Kincade ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Side Population ◽  
Expression Profiles ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.

scholarly journals The Ets2 Repressor Factor (Erf) Is Required for Effective Primitive and Definitive Hematopoiesis

2017 ◽  
Vol 37 (19) ◽  
Author(s):  
Ioanna Peraki ◽  
James Palis ◽  
George Mavrothalassitis
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Blood Cells ◽  
Fetal Liver ◽  
Normal Function ◽  
Severe Anemia ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Definitive Hematopoiesis ◽  
Cell Maintenance

ABSTRACT Erf is a gene for a ubiquitously expressed Ets DNA-binding domain-containing transcriptional repressor. Erf haploinsufficiency causes craniosynostosis in humans and mice, while its absence in mice leads to failed chorioallantoic fusion and death at embryonic day 10.5 (E10.5). In this study, we show that Erf is required in all three waves of embryonic hematopoiesis. Mice lacking Erf in the embryo proper exhibited severe anemia and died around embryonic day 14.5. Erf epiblast-specific knockout embryos had reduced numbers of circulating blood cells from E9.5 onwards, with the development of severe anemia by E14.5. Elimination of Erf resulted in both reduced and more immature primitive erythroblasts at E9.5 to E10.5. Reduced definitive erythroid colony-forming activity was found in the bloodstream of E10.5 embryos and in the fetal liver at E11.5 to E13.5. Finally, elimination of Erf resulted in impaired repopulation ability, indicating that Erf is necessary for hematopoietic stem cell maintenance or differentiation. We conclude that Erf is required for both primitive and erythromyeloid progenitor waves of hematopoietic stem cell (HSC)-independent hematopoiesis as well as for the normal function of HSCs.

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