Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

Download Full-text

Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver

Blood ◽

10.1182/blood.v96.12.3757.h8003757_3757_3762 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3757-3762 ◽

Cited By ~ 1

Author(s):

Hsiang-Chun Hsu ◽

Hideo Ema ◽

Mitsujiro Osawa ◽

Yukio Nakamura ◽

Toshio Suda ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Receptor Tyrosine Kinase ◽

Fetal Liver ◽

Newborn Mice ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Tie-2 receptor tyrosine kinase expressed in endothelial and hematopoietic cells is believed to play a role in both angiogenesis and hematopoiesis during development of the mouse embryo. This article addressed whether Tie-2 is expressed on fetal liver hematopoietic stem cells (HSCs) at day 14 of gestation. With the use of anti–Tie-2 monoclonal antibody, its expression was detected in approximately 7% of an HSC population of Kit-positive, Sca-1–positive, lineage-negative or -low, and AA4.1-positive (KSLA) cells. These Tie-2–positive KSLA (T+ KSLA) cells represent 0.01% to 0.02% of fetal liver cells. In vitro colony and in vivo competitive repopulation assays were performed for T+ KSLA cells and Tie-2–negative KSLA (T− KSLA) cells. In the presence of stem cell factor, interleukin-3, and erythropoietin, 80% of T+ KSLA cells formed colonies in vitro, compared with 40% of T− KSLA cells. Long-term multilineage repopulating cells were detected in T+ KSLA cells, but not in T− KSLA cells. An in vivo limiting dilution analysis revealed that at least 1 of 8 T+ KSLA cells were such repopulating cells. The successful secondary transplantation initiated with a limited number of T+ KSLA cells suggests that these cells have self-renewal potential. In addition, engraftment of T+ KSLA cells in conditioned newborn mice indicates that these HSCs can be adapted equally by the adult and newborn hematopoietic environments. The data suggest that T+ KSLA cells represent HSCs in the murine fetal liver.

Download Full-text

Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver

Blood ◽

10.1182/blood.v96.12.3757 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3757-3762 ◽

Cited By ~ 47

Author(s):

Hsiang-Chun Hsu ◽

Hideo Ema ◽

Mitsujiro Osawa ◽

Yukio Nakamura ◽

Toshio Suda ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Receptor Tyrosine Kinase ◽

Fetal Liver ◽

Newborn Mice ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Tie-2 receptor tyrosine kinase expressed in endothelial and hematopoietic cells is believed to play a role in both angiogenesis and hematopoiesis during development of the mouse embryo. This article addressed whether Tie-2 is expressed on fetal liver hematopoietic stem cells (HSCs) at day 14 of gestation. With the use of anti–Tie-2 monoclonal antibody, its expression was detected in approximately 7% of an HSC population of Kit-positive, Sca-1–positive, lineage-negative or -low, and AA4.1-positive (KSLA) cells. These Tie-2–positive KSLA (T+ KSLA) cells represent 0.01% to 0.02% of fetal liver cells. In vitro colony and in vivo competitive repopulation assays were performed for T+ KSLA cells and Tie-2–negative KSLA (T− KSLA) cells. In the presence of stem cell factor, interleukin-3, and erythropoietin, 80% of T+ KSLA cells formed colonies in vitro, compared with 40% of T− KSLA cells. Long-term multilineage repopulating cells were detected in T+ KSLA cells, but not in T− KSLA cells. An in vivo limiting dilution analysis revealed that at least 1 of 8 T+ KSLA cells were such repopulating cells. The successful secondary transplantation initiated with a limited number of T+ KSLA cells suggests that these cells have self-renewal potential. In addition, engraftment of T+ KSLA cells in conditioned newborn mice indicates that these HSCs can be adapted equally by the adult and newborn hematopoietic environments. The data suggest that T+ KSLA cells represent HSCs in the murine fetal liver.

Download Full-text

Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential

Blood ◽

10.1182/blood-2008-12-192054 ◽

2009 ◽

Vol 113 (25) ◽

pp. 6342-6350 ◽

Cited By ~ 205

Author(s):

David G. Kent ◽

Michael R. Copley ◽

Claudia Benz ◽

Stefan Wöhrer ◽

Brad J. Dykstra ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Self Renewal ◽

Hematopoietic Stem ◽

New Genes ◽

Essential Components

Abstract Hematopoietic stem cells (HSCs) are generally defined by their dual properties of pluripotency and extensive self-renewal capacity. However, a lack of experimental clarity as to what constitutes extensive self-renewal capacity coupled with an absence of methods to prospectively isolate long-term repopulating cells with defined self-renewal activities has made it difficult to identify the essential components of the self-renewal machinery and investigate their regulation. We now show that cells capable of repopulating irradiated congenic hosts for 4 months and producing clones of cells that can be serially transplanted are selectively and highly enriched in the CD150+ subset of the EPCR+CD48−CD45+ fraction of mouse fetal liver and adult bone marrow cells. In contrast, cells that repopulate primary hosts for the same period but show more limited self-renewal activity are enriched in the CD150− subset. Comparative transcriptome analyses of these 2 subsets with each other and with HSCs whose self-renewal activity has been rapidly extinguished in vitro revealed 3 new genes (VWF, Rhob, Pld3) whose elevated expression is a consistent and selective feature of the long-term repopulating cells with durable self-renewal capacity. These findings establish the identity of a phenotypically and molecularly distinct class of pluripotent hematopoietic cells with lifelong self-renewal capacity.

Download Full-text

Murine Hematopoietic Stem Cells Require Akt1 and Akt2 for Long-Term Self-Renewal

Blood ◽

10.1182/blood.v112.11.502.502 ◽

2008 ◽

Vol 112 (11) ◽

pp. 502-502

Author(s):

Marisa M. Juntilla ◽

Vineet Patil ◽

Rohan Joshi ◽

Gary A. Koretzky

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Akt Signaling ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Murine hematopoietic stem cells (HSCs) rely on components of the Akt signaling pathway, such as FOXO family members and PTEN, for efficient self-renewal and continued survival. However, it is unknown whether Akt is also required for murine HSC function. We hypothesized that Akt would be required for HSC self-renewal, and that the absence of Akt would lead to hematopoietic failure resulting in developmental defects in multiple lineages. To address the effect of Akt loss in HSCs we used competitive and noncompetitive murine fetal liver-bone marrow chimeras. In short-term assays, Akt1−/−Akt2−/− fetal liver cells reconstituted the LSK compartment of an irradiated host as well or better than wildtype cells, although failed to generate wildtype levels of more differentiated cells in multiple lineages. When placed in a competitive environment, Akt1−/−Akt2−/− HSCs were outcompeted by wildtype HSCs in serial bone marrow transplant assays, indicating a requirement for Akt1 and Akt2 in the maintainance of long-term hematopoietic stem cells. Akt1−/−Akt2−/− LSKs tend to remain in the G0 phase of the cell cycle compared to wildtype LSKs, suggesting the failure in serial transplant assays may be due to increased quiesence in the absence of Akt1 and Akt2. Additionally, the intracellular content of reactive oxygen species (ROS) in HSCs is dependent on Akt signaling because Akt1−/−Akt2−/− HSCs have decreased ROS levels. Furthermore, pharmacologic augmentation of ROS in the absence of Akt1 and Akt2 results in an exit from quiescence and rescue of differentiation both in vivo and in vitro. Together, these data implicate Akt1 and Akt2 as critical regulators of long-term HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

Download Full-text

A Novel Conditional Mouse Model For MLL-ENL Induced Acute Leukemia

Blood ◽

10.1182/blood.v122.21.1277.1277 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1277-1277

Author(s):

Vaia Stavropoulou ◽

Sabine Juge ◽

Alexander Tzankov ◽

Michael Kyba ◽

Antoine H.F.M. Peters ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Acute Leukemia ◽

Hematopoietic Stem Cells ◽

Fusion Gene ◽

Mixed Lineage Leukemia ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The t(11;19) translocation leading to the MLL-ENL fusion is recurrently found in pediatric and adult de novo and therapy related mixed-lineage acute leukemia and is often associated with a poor prognosis. Previous studies have shown that (retroviral) overexpression of MLL-ENL potently immortalizes bone marrow cells in vitro and induces a lethal acute myeloid leukemia (AML) in mice. To establish a mouse model that phenocopies more closely the human disease, we generated conditional transgenic mice in which the expression of MLL-ENL is controlled by doxycycline (DOX) through a stably integrated reverse tet-responsive transactivator (rtTA). Induction of MLL-ENL expression in newborn or adult mice resulted in a leukemic phenotype that phenocopied pre-B- and myeloid mixed lineage leukemia as observed in most patients with MLL-ENL. The diseased mice displayed excessive splenomegaly, massive lymph node as well as multiple organ infiltration by two co-existing types of blasts mostly expressing higher or lower levels of B220 and Gr1/Mac1 and similar levels of c-kit. Expression of the fusion gene and disease induction was DOX dosage dependent and reversible upon DOX removal. Despite significantly lower fusion gene expression levels as we observed in retroviral systems the median latency for the development of the disease in this model (104.3±16.9 days) was comparable to them (62±10.4 days) and much shorter than any of the previously reported MLL-ENL knock-in mouse models (> 1 year). Continuous ex vivo expression of MLL-ENL provided bone marrow and fetal liver hematopoietic cells with a strong self-renewal capacity and caused the accumulation of immature blast-like cells upon serial replating in methylcellulose cultures. In the presence of factors favoring myelopoiesis, like IL-3, DOX removal resulted in a complete differentiation towards the granulocytic-monocytic, lineages expressing high levels of Mac1/Gr-1, whereas IL-7 favored differentiation towards the B-cell lineage characterized by the expression of high levels of B220. In addition, MLL-ENL induced a DOX dependent aberrant self renewal capacity and a differentiation block in methylcellulose cultures of hematopoietic stem cells (Lin- c-kit+ Sca-1+, LSK) and various progenitors including common lymphoid progenitor (CLP) and granulocyte-macrophage progenitor (GMP) cells. Interestingly, MLL-ENL expression preferentially expanded LSK- rather than GMP-derived cells as assayed by growth curves in long-term (> 1 month) liquid cultures in the presence of cytokines in vitro. In line with this observation, in vivo, expression of MLL-ENL in long-term hematopoietic stem cells (LT-HSC) induced an aggressive mixed lineage leukemia characterized by the presence of two distinguishable populations of blasts, whereas induction in GMPs never induced a disease. These data suggest that MLL-ENL preferentially transforms hematopoietic stem cells rather than more differentiated progenitors. Thus, this novel transgenic mouse model for MLL-ENL induced acute leukemia closely recapitulates the human disease and combines the advantages of the existing knock-in and retroviral models. This model allowed us to demonstrate that in contrast to the MLL-AF9 fusion, that preferentially immortalizes GMPs, MLL-ENL preferentially transforms HSCs. We anticipate that our model will be a valuable tool to study the cellular origin and to search for and/or validate novel therapeutic targets for MLL-ENL induced acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Faculty Opinions recommendation of Cultivation of aorta-gonad-mesonephros-derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004105.47154 ◽

2002 ◽

Author(s):

Ana Cumano

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Hematopoietic Stem

Download Full-text

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

Download Full-text

Repopulating Kupffer Cells Originate Directly from Hematopoietic Stem Cells

10.21203/rs.3.rs-1034533/v1 ◽

2021 ◽

Author(s):

Xu Fan ◽

Pei Lu ◽

Xianghua Cui ◽

Peng Wu ◽

Weiran Lin ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Kupffer Cells ◽

Lineage Tracing ◽

Genetic Lineage ◽

Fate Mapping ◽

Monocytic Cell ◽

Hematopoietic Stem ◽

Cellular Origin

Abstract Kupffer cells (KCs) originate from yolk sac progenitors before birth, but the origin of repopulating KCs in adult remains unclear. In current study, we firstly traced the fate of preexisting KCs and that of monocytic cells with tissue-resident macrophage-specific and monocytic cell-specific fate mapping mouse models, respectively, and found no evidences that repopulating KCs originate from preexisting KCs or MOs. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC depletion in mice, and found that in response to KC depletion, hematopoietic stem cells (HSCs) proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Finally, we traced the fate of HSCs in a HSC-specific fate-mapping mouse model, in context of chronic liver inflammation induced by repeated carbon tetrachloride treatment, and confirmed that repopulating KCs originated directly from HSCs. Taken together, these findings provided in vivo fate-mapping evidences that repopulating KCs originate directly from hematopoietic stem cells, which present a completely novel understanding of the cellular origin of repopulating Kupffer Cells and shedding light on the divergent roles of KCs in liver homeostasis and diseases.

Download Full-text

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v94.5.1623 ◽

1999 ◽

Vol 94 (5) ◽

pp. 1623-1636 ◽

Cited By ~ 72

Author(s):

Chu-Chih Shih ◽

Mickey C.-T. Hu ◽

Jun Hu ◽

Jeffrey Medeiros ◽

Stephen J. Forman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

In Vitro Culture ◽

Culture System ◽

Ex Vivo ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

Download Full-text