The effect of 5-fluorouracil on erythropoiesis

Abstract The effects of a single dose (150 mg/kg) of 5-fluorouracil on mature erythroid and erythropoietic and multipotential in vitro precursor populations in the bone marrow and spleen and circulating biologically (erythroid colony forming unit [CFU-E] assay) and immunologically active (enzyme-linked immunosorbent assay) erythropoietin (Epo) are described. All mature erythroid (reticulocytes, erythrocytes) and in vitro erythropoietic precursors (CFU-E, erythroid burst-forming unit [BFU-E]) are severely reduced, if not eradicated. Transient repopulation of the pure BFU-E and CFU-E populations on days 6 and 7, respectively, produces a marked reticulocytosis after day 9. Circulating Epo increases to above normal values by day 2. However, whereas biologically active Epo remains constant at this level until day 9, immunologically active Epo continually increases; by day 12, however, both assays detect circulating Epo levels of about 400 mU/mL. In vitro multipotential stem cells (BFU-E mix) are reduced to 32% on day 1, 7.6% on day 2, and return to normal values between days 4 and 5. The survival and repopulation kinetics of the BFU-E mix imply a stem cell population more mature than the high proliferative potential colony-forming cells. However, the BFU-E mix may be responsible for erythropoiesis repopulating ability.

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The effect of 5-fluorouracil on erythropoiesis

Blood ◽

10.1182/blood.v77.6.1164.bloodjournal7761164 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1164-1170

Author(s):

IN Rich

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Normal Values ◽

Biologically Active ◽

Stem Cell Population ◽

Enzyme Linked Immunosorbent Assay ◽

Proliferative Potential ◽

Colony Forming Unit ◽

Kinetics Of

The effects of a single dose (150 mg/kg) of 5-fluorouracil on mature erythroid and erythropoietic and multipotential in vitro precursor populations in the bone marrow and spleen and circulating biologically (erythroid colony forming unit [CFU-E] assay) and immunologically active (enzyme-linked immunosorbent assay) erythropoietin (Epo) are described. All mature erythroid (reticulocytes, erythrocytes) and in vitro erythropoietic precursors (CFU-E, erythroid burst-forming unit [BFU-E]) are severely reduced, if not eradicated. Transient repopulation of the pure BFU-E and CFU-E populations on days 6 and 7, respectively, produces a marked reticulocytosis after day 9. Circulating Epo increases to above normal values by day 2. However, whereas biologically active Epo remains constant at this level until day 9, immunologically active Epo continually increases; by day 12, however, both assays detect circulating Epo levels of about 400 mU/mL. In vitro multipotential stem cells (BFU-E mix) are reduced to 32% on day 1, 7.6% on day 2, and return to normal values between days 4 and 5. The survival and repopulation kinetics of the BFU-E mix imply a stem cell population more mature than the high proliferative potential colony-forming cells. However, the BFU-E mix may be responsible for erythropoiesis repopulating ability.

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111 EQUINE AMNIOTIC EPITHELIAL OR BONE MARROW MESENCHYMAL STEM CELLS DIFFERENTLY SUPPORT IN VITRO EMBRYO DEVELOPMENT IN A BOVINE IN VITRO CULTURE MODEL

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab111 ◽

2009 ◽

Vol 21 (1) ◽

pp. 156 ◽

Cited By ~ 1

Author(s):

F. Cremonesi ◽

V. Maggio ◽

A. Lange Consiglio

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

In Vitro Culture ◽

Calf Serum ◽

Biologically Active ◽

Bovine Embryo ◽

Epithelial Stem Cells ◽

Marrow Mesenchymal Stem Cells

There are indications that the culture system and the medium composition can affect embryo quality. In fact, various studies have been shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In light of this, recently, some studies used co-culture with mouse embryonic fibroblasts in the effort to improve the development of bovine and ovine in vitro-derived embryos. Despite the progress in equine IVM and ICSI technologies and the different culture conditions reported for preimplantation development of ICSI fertilized horse oocytes, the yield of blastocysts remained low. In the present study we investigated the benefits of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSC) or equine amniotic epithelial stem cells (AE-SC) on blastocyst development. This study employed the bovine embryo as a model and represents the initial step towards standardization of a protocol for the culture of equine embryos in our laboratory. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and layered over Hystopaque™ 1.080, then centrifuged for 20 min at 400g and 4°C. Cell pellets were resuspended in 10 mL Dulbecco Modified Earle’s Medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, penicillin (100 U mL–1) and streptomycin (100 μg mL–1) and seeded in 24-well plates. Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fractions were incubated at 37°C with 0.05% trypsin for 45 min. Separated AE cells were plated on 25 cm2 flask in standard culture media containing 10 ng mL–1 epidermal growth factor. Seven hundred fifty cumulus–oocyte complexes with a homogeneous cytoplasm and two or more layers of cumulus cells were used. After IVM and IVF cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured for up to Day 7: 1) co-culture with granulosa cells (control); 2) co-culture with BM-MSC; 3) co-culture with AE-SC. The culture medium was TCM 199 + 10% fetal bovine serum, pyruvate and gentamicin at 38.5°C in 5% CO2. Statistical analyses was performed by chi square test. Blastocysts developmental rates were similar among control, AE-SC and BM-MSC (35%, 41% and 30%, respectively), but the co-culture with AE-SC gave a significantly greater percentage of blastocysts compared to BM-MSC (P < 0.05). In conclusion, despite the absence of a significant increment in blastocysts attainment using stem cells as feeders for embryo culture, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSC. It can be suggested that these cells secrete biologically active substances including signaling molecules and growth factors of epithelial nature different from those of the BM cells of mesenchymal origin. Regione Lombardia is acknowledged for the “Dote Ricercatori” fellowship to V.M.

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Interleukin-11 stimulates multilineage progenitors, but not stem cells, in murine and human long-term marrow cultures

Blood ◽

10.1182/blood.v86.1.128.bloodjournal861128 ◽

1995 ◽

Vol 86 (1) ◽

pp. 128-134 ◽

Cited By ~ 18

Author(s):

XX Du ◽

D Scott ◽

ZX Yang ◽

R Cooper ◽

XL Xiao ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Fold Increase ◽

Proliferative Potential ◽

Colony Forming Unit ◽

Bone Marrow Cultures ◽

Progenitor Compartment ◽

Interleukin 11 ◽

Marrow Cultures

Interleukin-11 (IL-11) is a bone marrow microenvironment-derived growth factor with pleiotropic effects on a variety of hematopoietic cells. To more accurately assess the effects of IL-11 on stem and progenitor compartments within the hematopoietic microenvironment (HM), we added recombinant human (rh) IL-11 to human and murine long-term bone marrow cultures (LTMC) and analyzed primitive (high proliferative potential- colony forming cells [HPP-CFC], long-term culture-initiating cells [LTC- IC], and long-term reconstituting stem cells) and progenitor (day 12 colony forming unit-spleen [CFU-S12], colony forming unit-megakaryocyte [CFU-Mk] and colony forming unit-granulocyte/macrophage [CFU-GM]) compartments throughout the duration of the cultures. rhIL-11 (100 ng/mL) added twice weekly resulted in significantly increased nonadherent (NA) cellularity, CFU-GM, and CFU-Mk production in human LTMC. Addition of rhIL-11 to murine LTMC was associated with a 5- to 40- fold increase in CFU-GM and a four- to 20-fold increase in day 12 CFU-S in NA cells. However, IL-11 had no significant effect on total HPP-CFC concentration and decreased the size of the more primitive stem/progenitor compartment as evidenced by both decreased LTC-IC frequency in human LTMC and decreased frequency of long-term reconstituting stem cells in murine LTMC. These data suggest that IL-11 may increase commitment of stem cells into a multipotential progenitor compartment.

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Bone marrow stromal cells induce myeloid and lymphoid development of the sorted hematopoietic stem cells in vitro

Blood ◽

10.1182/blood.v86.7.2590.2590 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2590-2597 ◽

Cited By ~ 17

Author(s):

R Okuyama ◽

M Koguma ◽

N Yanai ◽

M Obinata

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Stromal Cell ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Stem Cell Population ◽

Hematopoietic Stem

Abstract Regulation of development of hematopoietic stem cells was examined by culturing Lin- c-Kit+ Sca1+ stem cells sorted from bone marrow (BM) cells by fluorescence-activated cell sorting on a layer of TBR59, a BM stromal cell line established from simian virus 40 T-antigen gene transgenic mice. The sorted stem cells did not show self-renewal, but two waves (at 7 and 13 days) of a cobblestone formation were induced by the stromal cell layer. The cobblestones were formed by finite cell division (eight divisions on average) of sorted Lin-c-Kit+ Sca1+ stem cells, and divided cells were still immature. The c-Kithigh stem cell population was induced to form the first wave of cobblestone formation committed to myeloid lineage, whereas c-Kitlow population was induced to form the second wave of this formation committed to lymphoid lineage. Both cobblestone formations require c-Kit function, but very late activation antigen-4-vascular cell adhesion molecule-1 interaction plays different parts in the two lineages.

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ROS-Mediated Iron Overload Injures The Hematopoiesis Of Bone Marrow By Damaging Hematopoietic Stem/Progenitor Cells In Mice

Blood ◽

10.1182/blood.v122.21.962.962 ◽

2013 ◽

Vol 122 (21) ◽

pp. 962-962

Author(s):

Xiao Chai ◽

Deguan Li ◽

Mingfeng Zhao ◽

Wenyi Lu ◽

Juan Mu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Iron Overload ◽

Normal Control ◽

Mice Model ◽

Inhibitory Effects ◽

Hematopoietic Stem ◽

Colony Forming Unit ◽

Iron Deposits

Abstract A substantial portion of patients with inherited blood disorders such as beta thalassemia, or bone marrow failure syndromes such as aplastic anemia(AA), myelodysplastic syndromes(MDS) require frequent transfusions of red blood cells. Frequent blood transfusions may lead to the excess of plasma non-transferrin -bound iron(NTBI) and iron overload occurs, which will significantly injure bone marrow (BM) function as well as induce organ dysfunctions such as liver cirrhosis, diabetes and cardiac diseases. However, the exact mechanism behind this effect remains elusive and ideal treatment needs to be explored. In our preliminary studies, we have demonstrated free iron catalyzes oxidative damage to hematopoietic cells/ mesenchymal stem cells in vitro and suppresses hematopoiesis in iron overload patients (Zhao et al.,blood, 2010 abstract; Lu et al.,blood,2012 abstract; Lu et al., Eur J Haematol, 2013). Here we observed the hematopoiesis inhibitory effects of iron overload on the basis of estabalished iron overload mice model and preliminarily disscussed the mechanism. In this study, we first established an iron overload mice model by administering different doses(12.5mg/ml,25mg/ml,50mg/ml) iron dextran by intraperitoneal injection every three days for four weeks. To confirm the efficacy of the mice model, the BM, hepatic and splenic iron deposits were observed by morphological study and the labile iron pool level (LIP) of bone marrow mononuclear cells(BMMNCs) was detected using the calcein-AM fluorescent dye. It was found that iron deposits in BM cells of iron overload mice, liver and spleen were markedly increased and the BMMNCs LIP level was much higher than that of normal control mice. The above results showed that the iron-overloaded mice model has been established successfully. Next we observed whether iron overload (25mg/ml) could affect the hematopoiesis of BM. The colony-forming cell assay was performed by culturing BMMNCs in MethoCult M3434 methylcellulose medium to evaluate hematopoietic progenitor cells(HPCs) proliferation function. The competitive repopulation assay and single-cell colony cultures of sorted hematopoietic stem cells (HSCs,CD34-Lin- sca1+c-kit+cells,LSK+)were used to validate HSCs function. The counts of BMMNCs have no significant difference. However, It was found that hematopoietic colony-forming unit (CFU-E, BFU-E, CFU-GM and CFU-mix) was much lower than that of normal control(P<0.05)(Fig.1). Notely, the number of LSK+ cells (*103/femur) was decreased significantly in iron overload mouse (26.43±3.28) compared with normal control(40.12±5.21) and the single-cell colony formation(/60wells) was reduced significantly in iron overload mouse(28.54±3.33) compared with normal control(47.93±4.82) (P<0.05). The long-term and multilineage engraftment capability of the iron-overloaded HSCs was weaken after transplantation. We then explored the possible mechanism of this inhibitory effects. Our previous studies have shown that iron overload could elevated reactive oxygen species (ROS) levels of mesenchymal stem cells and HSCs in vitro. Similarly, the intracellular ROS levels were analyzed by a flow cytometer. It was found that ROS level in iron overload BM was increased by 3.32 folds in erythroid cells, 1.51 folds in granulocytes and 4.80 folds in LSK+ cells,respectively. And also, the expression of p53, p38MAPK and p16Ink4a mRNA remained significantly elevated, which indicated that ROS related signal pathway was involved in the deficient hematopoiesis of iron overload BM. In addition, we also observed the effects of iron overload on the mice with deficient hematopoiesis exposed to 4Gy total body irradiation(TBI), which was more similar to clinical pathological conditions such as AA or MDS. It was found that BM damage caused by iron overload was aggravated in pathological conditions (primary findings were not shown). In conclusion, our study confirmed that iron overload injures the hematopoiesis of BM by enhancing oxidative stress in mice, which would be helpful to further study on the mechanism and would provide an experimental basis to find new therapeutic targets for the treatment of iron overload in patients with hematopoietic dysfunction.Figure 1Results of hematopoietic colony forming unit of different groups(*P<0.05)Figure 1. Results of hematopoietic colony forming unit of different groups(*P<0.05) Disclosures: No relevant conflicts of interest to declare.

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Bone marrow stromal cells induce myeloid and lymphoid development of the sorted hematopoietic stem cells in vitro

Blood ◽

10.1182/blood.v86.7.2590.bloodjournal8672590 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2590-2597

Author(s):

R Okuyama ◽

M Koguma ◽

N Yanai ◽

M Obinata

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Stromal Cell ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Stem Cell Population ◽

Hematopoietic Stem

Regulation of development of hematopoietic stem cells was examined by culturing Lin- c-Kit+ Sca1+ stem cells sorted from bone marrow (BM) cells by fluorescence-activated cell sorting on a layer of TBR59, a BM stromal cell line established from simian virus 40 T-antigen gene transgenic mice. The sorted stem cells did not show self-renewal, but two waves (at 7 and 13 days) of a cobblestone formation were induced by the stromal cell layer. The cobblestones were formed by finite cell division (eight divisions on average) of sorted Lin-c-Kit+ Sca1+ stem cells, and divided cells were still immature. The c-Kithigh stem cell population was induced to form the first wave of cobblestone formation committed to myeloid lineage, whereas c-Kitlow population was induced to form the second wave of this formation committed to lymphoid lineage. Both cobblestone formations require c-Kit function, but very late activation antigen-4-vascular cell adhesion molecule-1 interaction plays different parts in the two lineages.

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Comparison of the Biological and Functional Characteristics of Mesenchymal Stem Cells From Intrahepatic and Identical Bone Marrow

10.21203/rs.3.rs-89980/v1 ◽

2020 ◽

Author(s):

Hongyu Zhang ◽

Jiejuan Lai ◽

Shifang Jiang ◽

Ling Shuai ◽

Yujun Zhang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Conditioned Medium ◽

Cell Population ◽

Neutralizing Antibodies ◽

Stem Cell Population ◽

Functional Characteristics ◽

Fibrotic Liver

Abstract Background: We previously isolated a novel mesenchymal stem cell (MSC)-like neuroglial antigen 2-expressing stem cell population (MLpvNG2) from an uninjured liver by using a “Porcoll-Plate-Wait” method and determined that MLpvNG2 possesses hepatic stem/progenitor cell characteristics.Methods: In this study, we compared the biological and functional characteristics of the intrahepatic (MSC)-like MLpvNG2 with identical bone marrow-derived MSCs (niBM-MSCs), which are a well-accepted stem cell population in the field. We performed an in vitro study using conditioned medium and in vivo study using our well-set diethylnitrosamine (DEN)-induced liver fibrotic/cirrhotic murine model as well. Results: We found that in a fibrotic liver environment, MLpvNG2 survived better than niBM-MSCs obtained through different mechanisms of action. MLpvNG2 mainly differentiates into albumin (ALB(+)) hepatocytes, while niBM-MSCs mainly differentiate into CK/KRT19(+) cholangiocytes. Of note, we identified for the first time that C/EBPα/β is expressed on the cell surface of donor and host hepatic cells. As such, we used anti-C/EBPs neutralizing antibodies to determine the functional characteristics both in vitro (conditioned medium) and in the DEN-induced animal model.Conclusions: Based on our findings, it can be concluded that native-source (liver) stem cells (MLpvNG2) are more efficient than nonnative-sourced stem cells (niBM-MSCs) in the treatment of native (liver)-sourced diseases such as end-stage liver disease.

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EXPERIMENTAL SUBSTANTIATION OF THE USE OF MARAL DEER ANTLERS FOR COMBATING EXTREME PSYCHO-EMOTIONAL STRESS

Biomeditsina ◽

10.33647/2074-5982-15-3-33-40 ◽

2019 ◽

pp. 33-40

Author(s):

I. N. Smirnova ◽

N. I. Suslov ◽

I. A. Khlusov ◽

K. V. Zaytsev ◽

A. A. Gostyukhina ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Sleep Deprivation ◽

Hematopoietic Stem Cells ◽

Biologically Active ◽

Distilled Water ◽

Hematopoietic Stem ◽

In Vivo Experiments

This work was aimed at investigating the effect of maral antler powder on the activity of animal hematopoietic stem cells both in vivo and in vitro.For in vivo experiments based on the model of sleep deprivation, male mice of the CBA/CaLac line were used. Prior to the experiment, mice in the experimental and control groups were intragastrically administered with a water dispersion of a maral antler powder and distilled water, respectively. Subsequently, the extraction of bone marrow from the femur, cloning of erythro- and granulo-monocytopoiesis precursors and count of the number of cell colonies were performed. Experiments in vitro involved the extraction of bone marrow cells from the femur followed by their cultivation both in a culture containing a maral antler powder (experimental) and distilled water (control culture). The number of CFU was counted 7 days following the beginning of the experiment.Maral antlers are found to exhibit no noticeable modulating effect on the colony-forming activity of mouse hematopoietic stem cells in vitro. However, according to our in vivo experiments on mice, a preventive administration of an antler powder before a stressful infl uence (sleep deprivation) prevents suppression of erythropoiaesis processes, thus exhibiting a modulating effect on the activity of CFU-E and CFU-GM by increasing the number of CFU-E and reducing the number of CFU-GM by more than three times. The modulating effect of maral antlers on the activity of hematopoietic and stem cells is based on the infl uence of biologically active substances contained therein on the neuroendocrine regulation of the hematopoietic system occurring in living organisms.

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The Bone Marrow "Mystery Population" of Stem Cells 20 Years Later - a Puzzle Solved?

Blood ◽

10.1182/blood.v126.23.2392.2392 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2392-2392

Author(s):

Malwina Suszynska ◽

Daniel Pedziwiatr ◽

Magdalena J Kucia ◽

Mariusz Z Ratajczak ◽

Janina Ratajczak

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Receptor Expression ◽

Stem Cell Population ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Low Levels

Abstract Background . Almost 20 years ago, a "mystery" population of small stem cells with many of the phenotypic characteristics attributed to resting hematopoietic stem cells was identified in murine bone marrow (BM) (Stem Cells 1998, 16, 38-48). These cells expressed high levels of Sca-1, H-2K, and CD38 and low levels of Thy-1.1; they expressed CD45 antigen but were lineage-negative (lin-) for other hematopoietic markers. These cells incorporated only low levels of Rh123 and were resistant to the cytotoxic effects of 5-fluorouracil. The only phenotypic characteristic that distinguishes these cells from Sca-1+, Lin-, CD45+ Thy-1.1low long-term-reconstituting hematopoietic stem cell population is the lack of c-kit expression. In sum, this "mystery" population of small Sca-1+, lin-, c-kit- but CD45+ stem cells do not respond to hematopoietic growth factors in vitro, form in vivo spleen colonies, or reconstitute lethally irradiated mice. With our discovery of Sca-1+ Lin- CD45- very small embryonic-like stem cells (VSELs) in murine bone marrow (BM) (Leukemia 2006, 20, 857-869), we became interested in this "mystery" population of stem cells. VSELs, like the "mystery" population, are c-kit - and, if freshly isolated from BM, do not show any hematopoietic activity in standard in vitro and in vivo assays. In order to become specified to hematopoiesis, they need to be expanded over an OP-9 stromal support (Exp Hematol 2011;39:225-237). Hypothesis. Since (1) very small CD45- VSELs can be specified in OP-9 co-cultures into long-term reconstituting CD45+ HSCs, (2) the size of the "mystery" population is intermediate between VSELs and HSCs, and (3) VSELs and HSCs differ in cell surface receptor expression, we hypothesized that the "mystery" population is a missing developmental intermediate between VSELs and HSCs. Materials and Methods . Multicolor FACS analysis was employed to compare size and expression of surface markers between murine BM HSCs, the unknown population of stem cells, and VSELs. Next, the populations of small Sca-1+ H2-K+ lin- c-kit+ CD38+/- CD45+ cells (HSCs), smaller Sca-1+ H-2K+ lin- c-kit- CD38+ CD45+ cells (the "mystery" population), and very small in size Sca-1+ H-2K+ lin- c-kit- CD38+/- CD45- cells (VSELs) were purified by FACS from BM (Figure 1) and tested for in vitro colony formation. All these cell populations were primed/expanded over OP-9 support and subsequently evaluated for their hematopoietic potential after passaging in consecutive methylocellulose cultures (passages 1-4). RQ-PCR analysis was employed for detection of pluripotency marker expression as well as hematopoietic gene expression. Results . We found that, in contrast to HSCs, neither freshly sorted stem cells from the "mystery" BM population nor, as expected, VSELs grew hematopoietic colonies in standard methylcellulose cultures. This was also an important step in excluding contamination of our sorted populations with clonogenic cells. We also found that, while VSELs highly expressed Oct-4, this transcription factor was expressed at very low levels in the "mystery" population and was not detectable in HSCs. The most important observation was that the "mystery" population of stem cells became specified in OP-9-supported cultures into clonogenic HSPCs, and this specification occurred faster than the delayed specification of VSELs. VSELs first became enriched for HSPCs after acquiring CD45 antigen expression. Conclusions . Based on the results presented, we propose that the "mystery" population in murine BM is a population of stem cells intermediate between the most primitive population of BM-residing stem cells (VSELs) and the population of stem cells already specified to lympho-hematopoietic development (HSCs). Disclosures No relevant conflicts of interest to declare.

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