scholarly journals Hemoglobin switching in sheep: characteristics of BFU-E-derived colonies from fetal liver

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 495-500
Author(s):  
JE Barker
Keyword(s):  
Stem Cells ◽  
Fetal Liver ◽  
Fetal Hemoglobin ◽  
Liver Cells ◽  
S Phase ◽  
Hemoglobin Switching ◽  
Fetal Liver Cells ◽  
Beta 2 ◽  
Number Of Cells

Two types of erythroid colonies were generated in vitro from sheep fetal liver cells. The first type consisted of single colonies of 8–256 cells that were well hemoglobinized by 4 days; these are thought to originate from CFU-E. The second type consisted of macroscopic colonies composed of several subcolonies that matured between days 3 and 8 in vitro. At maturity, each contained 256 to > 1000 cells that formed a discrete macroscopic cluster. The macroscopic colonies, not previously described in sheep, are thought to be derived from BFU-E. The characteristics of sheep BFU-E were defined and the production of fetal hemoglobin (HbF, alpha 1, gamma 2) and HbC (alpha 2 beta 2) was compared in colonies derived from CFU-E or BFU-E. Bursts developed at erythropoietin (epo) concentrations as low as 0.1 U/ml, although the number observed increased with epo concentration up to 10 U/ml. The number of bursts observed was approximately proportional to the number of cells plated. As shown by thymidine suicide, approximately 50% of both the BFU e and CFU-E were in S-phase when obtained from the fetus. BFU-E were smaller and partially separable from CFU-E after sedimentation at unit gravity. The beta c/gamma synthetic ratio in colonies derived from BFU-E was greater than in CFU-E-derived colonies. These data suggest that the capacity for generation of erythroblasts making HbC is greater in the earlier or more primitive erythroid stem cells in fetal liver.

scholarly journals Hemoglobin switching in sheep: characteristics of BFU-E-derived colonies from fetal liver

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 495-500 ◽  
Author(s):  
JE Barker
Keyword(s):  
Stem Cells ◽  
Fetal Liver ◽  
Fetal Hemoglobin ◽  
Liver Cells ◽  
S Phase ◽  
Hemoglobin Switching ◽  
Fetal Liver Cells ◽  
Beta 2 ◽  
Number Of Cells

Abstract Two types of erythroid colonies were generated in vitro from sheep fetal liver cells. The first type consisted of single colonies of 8–256 cells that were well hemoglobinized by 4 days; these are thought to originate from CFU-E. The second type consisted of macroscopic colonies composed of several subcolonies that matured between days 3 and 8 in vitro. At maturity, each contained 256 to > 1000 cells that formed a discrete macroscopic cluster. The macroscopic colonies, not previously described in sheep, are thought to be derived from BFU-E. The characteristics of sheep BFU-E were defined and the production of fetal hemoglobin (HbF, alpha 1, gamma 2) and HbC (alpha 2 beta 2) was compared in colonies derived from CFU-E or BFU-E. Bursts developed at erythropoietin (epo) concentrations as low as 0.1 U/ml, although the number observed increased with epo concentration up to 10 U/ml. The number of bursts observed was approximately proportional to the number of cells plated. As shown by thymidine suicide, approximately 50% of both the BFU e and CFU-E were in S-phase when obtained from the fetus. BFU-E were smaller and partially separable from CFU-E after sedimentation at unit gravity. The beta c/gamma synthetic ratio in colonies derived from BFU-E was greater than in CFU-E-derived colonies. These data suggest that the capacity for generation of erythroblasts making HbC is greater in the earlier or more primitive erythroid stem cells in fetal liver.

Impaired Repopulating Activity of Fus-Deficient Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2222-2222
Author(s):  
Takeaki Sugawara ◽  
Atsushi Iwama
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Chromosomal Translocation ◽  
Liver Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Fetal Liver Cells

Abstract RNA-binding protein FUS (also known as TLS) was originally identified in chromosomal translocation in human myxoid liposarcoma. The FUS gene is also translocated with the transcription factor gene ERG in human myeloid leukemia with recurrent chromosomal translocation t(16;21). Multiple data suggest that wild-type FUS is also involved in the development of leukemia as one of the downstream targets for oncoproteins including BCR-ABL. However, little is known about the role of FUS in the normal hematopoiesis. The previous report demonstrated that Fus-deficient (Fus−/−) newborn mice, which die shortly after birth because they cannot suckle, have a non-cell-autonomous defect in B lymphocyte development. No cell-autonomous defect of Fus−/− hematopoietic cells has been documented. Here we report the detailed analyses of the Fus−/− fetal liver hematopoietic stem cells (HSCs). Fus−/− fetal livers at embryonic day 14.5 were smaller in size and exhibited a significant reduction in hematopoietic cell numbers by 60% compared with the wild type (WT). Nonetheless, no significant difference was observed in the proportion of stem/progenitor cell fraction (lineage-marker-c-Kit+Sca-1+; KSL) as well as colony-forming cells between WT and Fus−/− fetal livers. Fus−/− KSL cells proliferated and differentiated almost normally in vitro. To examine in vivo repopulating activity, we transplanted fetal liver cells to lethally irradiated CD45.1 recipients with competitor bone marrow cells. Fus−/− fetal liver donor cells reconstituted recipients’ hematopoiesis for the long term and contributed to all cell lineages including B lymphocytes. In contrast to the in vitro results, however, the chimerism of donor-derived cells was significantly lower in recipients receiving Fus−/− fetal liver cells compared with WT controls (approximately 2-fold reduction). This trend was reproducible with both unfractionated and purified KSL fetal liver test cells. Our data demonstrated that the proto-oncogene Fus is involved in the maintenance of normal HSC functions. Detailed analyses on the underlying mechanisms are in progress.

The Erythropoietin Receptor Regulates The Number Of Cell Divisions and The Duration Of Erythroblast Terminal Differentiation By Regulating Erythroblast Iron

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 428-428
Author(s):  
Daniel Hidalgo ◽  
Ramona Pop ◽  
Prem Ponka ◽  
Merav Socolovsky
Keyword(s):  
Multispectral Imaging ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Terminal Differentiation ◽  
Liver Cells ◽  
S Phase ◽  
Erythropoietin Receptor ◽  
Cell Divisions ◽  
Fetal Liver Cells

Abstract Signaling by the erythropoietin receptor (EpoR) is essential for the survival of definitive colony-forming unit-erythroid (CFU-e) progenitors and their erythroblast progeny. Here we used EpoR-null embryos to ask whether EpoR signaling might also exert essential non-survival functions in erythropoiesis. To address this, we rescued EpoR-null fetal liver cells from death by transducing them in vitro with either the anti-apoptotic protein Bcl-xL, or, as control, with the wild-type EpoR. The Bcl-xL-transduced EpoR-null cells survived, expressed hemoglobin and underwent morphological erythroid maturation and enucleation. However, unlike exogenous EpoR, exogenous Bcl-xL was unable to support the formation of EpoR-null CFU-e colonies in methylcellulose. The absence of colonies was explained by the finding that the Bcl-xL-transduced progenitors underwent fewer cell divisions than equivalent EpoR-transduced cells (1.1 vs. 2.9 in 24 hr, respectively) and had a slower rate of intra-S phase DNA synthesis, suggesting longer S phase duration. Multispectral imaging showed that the Bcl-xL-transduced cells matured prematurely, attaining smaller cell and nuclear size and a lower nuclear/cytoplasmic ratio at earlier time points than EpoR-transduced cells. Premature maturation was also evident by flow cytometric analysis. Thus, EpoR-null fetal liver cells in vivo arrest in their differentiation at the transition from subset S0 (Ter119-neg CD71-low) to S1 (Ter119-neg CD71-high) (Pop et al, PLoS Biology 2010). Rescue with EpoR in vitro allows EpoR-null progenitors to resume differentiation, sequentially upregulating CD71 and Ter119. By contrast, rescue of EpoR-null cells with Bcl-xL results in their premature upregulation of Ter119 and failure to upregulate CD71 to high levels. The cell cycle and differentiation deficits in Bcl-xL-supported, EpoR-null erythropoiesis were associated with a slower loss of DNA methylation from the erythroid genome, and with slower erythroid gene transcription. CD71 (the transferrin receptor) is a known target of EpoR and Stat5 signaling. We asked whether the deficits of EpoR-null erythropoiesis might be the result of low cell surface CD71 and the consequent reduced iron transport. In support of this hypothesis, we found that EpoR-null fetal liver cells that are transduced with both CD71 and Bcl-xL resume the normal maturation rate characteristic of EpoR-supported differentiation, as judged by multispectral imaging measurements of cell size and nuclear/cytoplasmic ratio. Further, we were able to restore rapid S phase to Bcl-xL-transduced EpoR-/- erythroblasts by culturing them in the presence of the cell-permeant iron chelator Fe-SIH (salicylaldehyde isonicotinoyl hydrazone), which is able to supply the cell interior with iron even in the absence of CD71 (Figure 1). We suggest that EpoR-mediated upregulation of CD71 at the onset of erythroid terminal differentiation determines the number and duration of erythroblast cell divisions by regulating iron homeostasis. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic Defects in Cited2 Mutant Fetal Liver.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2272-2272
Author(s):  
Yu Chen ◽  
Yu-Chung Yang
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Cited2 [cAMP-responsive element-binding protein (CBP)/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is a newly identified transcriptional modulator. Knockout of Cited2 gene is embryonic lethal because of heart and neural tube defects. Cited2 binds directly to CBP and p300, which have been shown to be crucial for hematopoietic stem cell self-renewal and proper hematopoietic differentiation, respectively. Cited2 also induces the expression of a polycomb-group gene, Bmi-1, which is essential for self-renewal of adult hematopoietic stem cells. These connections provided rationale to study the potential role of Cited2 in hematopoiesis. Mouse fetal liver is the major hematopoietic organ from day 10 postcoitus until right before birth. The smaller sized Cited2−/− fetal liver and significantly decreased fetal liver cellularity strongly suggest the potential defect in hematopoiesis. In vitro colony formation assay in methycellulose-based medium was used to characterize the hematopoietic progenitors. We found that fetal liver cells from E13.5, 14.5 and E15.5 Cited2−/− embryos gave rise to much less colonies, which reflects the decreased number and proliferative ability of hematopoietic progenitors due to Cited2 deficiency. Immunostaining of lineage-specific cell surface markers followed by flow cytometry was performed to characterize different hematopoietic populations in E14.5 and E15.5 fetal liver of wild type and Cited2−/− embryos. Cited2−/− fetal liver cells displayed a significant reduction in numbers throughout the hematopoietic hierarchy including hematopoietic stem cells (Lin− c-Kit+ Sca-1+), progenitor cells (Lin− c-Kit+), and differentiated cells of different lineages (CD45+, Ter119+, Mac-1+, Gr-1+), thus revealing a multi-level hematopoietic deficiency of Cited2−/− embryos. Long-term reconstitution experiment was then carried out to measure the ability of hematopoietic stem cells from Cited2−/− fetal liver cells to engraft and reconstitute hematopoietic system of congenic recipient mice. Mice transplanted with Cited2−/− fetal liver cells showed reconstitution of T cells whereas a 2-fold decrease in the reconstitution of B cell and myeloid lineages was observed, indicating a compromised ability of Cited2−/− fetal liver hematopoietic stem cells to maintain hematopoiesis. The results suggest an important role of Cited2 in hematopoietic differentiation and a selective function of Cited2 in B lymphoid &myeloid induction. The underlying mechanisms responsible for these defects will be pursued by microarray analysis of gene expression profile of Cited2−/− fetal liver cells, followed by more detailed phenotypic analyses of B and myeloid lineage markers plus in vitro and in vivo functional assays.

scholarly journals Cultivation of Fetal Liver Cells in a Three-Dimensional Poly-L-Lactic Acid Scaffold in the Presence of Oncostatin M

2002 ◽  
Vol 11 (5) ◽  
pp. 403-406 ◽  
Author(s):  
Jinlan Jiang ◽  
Nobuhiko Kojima ◽  
Taisei Kinoshita ◽  
Atsushi Miyajima ◽  
Weiqun Yan ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Fetal Liver ◽  
Three Dimensional ◽  
Liver Cells ◽  
Oncostatin M ◽  
Albumin Secretion ◽  
Plla Scaffold ◽  
Fetal Liver Cells ◽  
Number Of Cells

To investigate the feasibility of fetal liver cells for liver tissue engineering, the supporting function of poly-l-lactic acid (PLLA) for fetal liver cells and the effects of oncostatin M (OSM) on hepatic differentiation were studied. After preparing three-dimensional biodegradable PLLA scaffold having a well-developed open-pore structure by a gas-forming method with ammonium chloride particles as a porogen and a gas-forming reagent, fetal liver cells separated from E14.5-C57BL/6CrSlc murine embryos were inoculated in the PLLA scaffolds. Cells were cultured in Williams' E medium with or without OSM (10 ng/ml) for 30 days with a medium change every 2 days. Results showed that there were significant increases in the number of cells and in albumin secretion in PLLA culture compared with in monolayer culture on day 15. In addition, a significant increase in albumin secretion was observed in OSM-added PLLA culture compared with OSM-free culture, and there was only a slightly enhanced albumin secretion in monolayer cultures with OSM. These results suggest that PLLA may enhance the biological activity of OSM for inducing maturation of fetal liver cells. Interestingly, the number of cells in PLLA culture with OSM decreased compared with OSM-free PLLA culture at day 15. This may be because promotion of hepatic development by OSM simultaneously suppressed in vitro hematopoiesis (i.e., blood cell production). In summary, our results indicate that the three-dimensional PLLA scaffold is a good support material for the cultivation of fetal liver cells and that OSM is capable of not only terminating hematopoiesis of the fetal liver but also stimulating the maturation of hepatic parenchymal cells in vitro.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF β-receptor null mice

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 1990-1998 ◽  
Author(s):  
Wolfgang E. Kaminski ◽  
Per Lindahl ◽  
Nancy L. Lin ◽  
Virginia C. Broudy ◽  
Jeffrey R. Crosby ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fetal Liver ◽  
Liver Cells ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Liver Growth ◽  
Hematologic Disorders ◽  
Fetal Liver Cells ◽  
Β Receptor

Abstract Platelet-derived growth factor (PDGF)-B and PDGF β-receptor (PDGFRβ) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B−/−, PDGFRβ−/−, or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFRβ expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses.

scholarly journals Separation of Erythropoietin-sensitive Cells From Hemopoietic Spleen Colony-forming Stem Cells of Mouse Fetal Liver by Unit Gravity Sedimentation

Blood ◽  
1971 ◽  
Vol 37 (4) ◽  
pp. 417-427 ◽  
Author(s):  
JOHN R. STEPHENSON ◽  
ARTHUR A. AXELRAD
Keyword(s):  
Stem Cells ◽  
Present Method ◽  
Fetal Liver ◽  
Sedimentation Velocity ◽  
Assay Method ◽  
Fetal Liver Cells ◽  
Gravity Sedimentation ◽  
Cell Fractions ◽  
Unit Gravity Sedimentation

Abstract An assay method for erythropoietin-sensitive cells has been developed based on the fact that cells of mouse fetal liver respond to erythropoietin in vitro by increased heme synthesis. To determine whether or not hemopoietic colony-forming stem cells are identical with erythropoietin-sensitive cells, C3H/Bi 13-day fetal liver cells were separated into fractions on the basis of size by unit gravity sedimentation through a 1-2 per cent bovine serum albumin gradient. The cell fractions obtained were assayed for erythropoietin-sensitive cells by the present method and for spleen colony-forming cells by the method of Till and McCulloch. It was found that the modal sedimentation velocity of erythropoietin-sensitive cells was greater than that of the spleen colony-forming cells of mouse fetal liver, showing that these two classes of cells are distinct.

scholarly journals Expansion of hematopoietic stem cells in the developing liver of a mouse embryo

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2284-2288 ◽  
Author(s):  
Hideo Ema ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mouse Embryo ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Cell Number ◽  
Liver Cells ◽  
Single Wave ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 × 105 adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential.

Disruption of palladin leads to defects in definitive erythropoiesis by interfering with erythroblastic island formation in mouse fetal liver

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 870-876 ◽  
Author(s):  
Xue-Song Liu ◽  
Xi-Hua Li ◽  
Yi Wang ◽  
Run-Zhe Shu ◽  
Long Wang ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Liver Cells ◽  
Normal Function ◽  
Yolk Sac ◽  
Neural Tube Closure ◽  
Island Formation ◽  
Erythroblastic Island ◽  
Fetal Liver Cells ◽  
Definitive Erythropoiesis

Abstract Palladin was originally found up-regulated with NB4 cell differentiation induced by all-trans retinoic acid. Disruption of palladin results in neural tube closure defects, liver herniation, and embryonic lethality. Here we further report that Palld−/− embryos exhibit a significant defect in erythropoiesis characterized by a dramatic reduction in definitive erythrocytes derived from fetal liver but not primitive erythrocytes from yolk sac. The reduction of erythrocytes is accompanied by increased apoptosis of erythroblasts and partial blockage of erythroid differentiation. However, colony-forming assay shows no differences between wild-type (wt) and mutant fetal liver or yolk sac in the number and size of colonies tested. In addition, Palld−/− fetal liver cells can reconstitute hematopoiesis in lethally irradiated mice. These data strongly suggest that deficient erythropoiesis in Palld−/− fetal liver is mainly due to a compromised erythropoietic microenvironment. As expected, erythroblastic island in Palld−/− fetal liver was found disorganized. Palld−/− fetal liver cells fail to form erythroblastic island in vitro. Interestingly, wt macrophages can form such units with either wt or mutant erythroblasts, while mutant macrophages lose their ability to bind wt or mutant erythroblasts. These data demonstrate that palladin is crucial for definitive erythropoiesis and erythroblastic island formation and, especially, required for normal function of macrophages in fetal liver.

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

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3757-3762 ◽  
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.

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