hGATA1 Under the Control of a μLCR/β-Globin Promoter Rescues the Erythroid but Not the Megakaryocytic Phenotype Induced by the Gata1low Mutation in Mice

The phenotype of mice carrying the Gata1low mutation that decreases expression of Gata1 in erythroid cells and megakaryocytes, includes anemia, thrombocytopenia, hematopoietic failure in bone marrow and development of extramedullary hematopoiesis in spleen. With age, these mice develop myelofibrosis, a disease sustained by alterations in stem/progenitor cells and megakaryocytes. This study analyzed the capacity of hGATA1 driven by a μLCR/β-globin promoter to rescue the phenotype induced by the Gata1low mutation in mice. Double hGATA1/Gata1low/0 mice were viable at birth with hematocrits greater than those of their Gata1low/0 littermates but platelet counts remained lower than normal. hGATA1 mRNA was expressed by progenitor and erythroid cells from double mutant mice but not by megakaryocytes analyzed in parallel. The erythroid cells from hGATA1/Gata1low/0 mice expressed greater levels of GATA1 protein and of α- and β-globin mRNA than cells from Gata1low/0 littermates and a reduced number of them was in apoptosis. By contrast, hGATA1/Gata1low/0 megakaryocytes expressed barely detectable levels of GATA1 and their expression of acetylcholinesterase, Von Willebrand factor and platelet factor 4 as well as their morphology remained altered. In comparison with Gata1+/0 littermates, Gata1low/0 mice contained significantly lower total and progenitor cell numbers in bone marrow while the number of these cells in spleen was greater than normal. The presence of hGATA1 greatly increased the total cell number in the bone marrow of Gata1low/0 mice and, although did not affect the total cell number of the spleen which remained greater than normal, it reduced the frequency of progenitor cells in this organ. The ability of hGATA1 to rescue the hematopoietic functions of the bone marrow of the double mutants was confirmed by the observation that these mice survive well splenectomy and did not develop myelofibrosis with age. These results indicate that hGATA1 under the control of µLCR/β-globin promoter is expressed in adult progenitors and erythroid cells but not in megakaryocytes rescuing the erythroid but not the megakaryocyte defect induced by the Gata1low/0 mutation.

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Campath-1H-Regulated Ex Vivo Expansion of Cord Blood: Selection of Stem Cells, Depletion of Lymphocytes and Preferential Expansion of Myeloid, Megakaryocytic and Erythroid Precursors.

Blood ◽

10.1182/blood.v108.11.3644.3644 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3644-3644

Author(s):

Feng Qi ◽

Che K. Lim ◽

Li Sun ◽

William Y.K. Hwang

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Cell Number ◽

Ex Vivo Expansion ◽

Total Cell ◽

Total Cell Number ◽

Erythroid Precursors ◽

Selection Of

Abstract Human umbilical cord blood (CB) represents a unique source of transplantable hematopoietic cells. Unfortunately, when the cell dose is less than optimal, delayed or failed engraftment may result. To overcome this limitation, ex vivo expansion of CB products in an attempt to increase the number of progenitor cells has been evaluated. However, current clinical conditions for ex vivo expansion of CB cells require selection of the CD34+ or CD133+ subset, as unfractionated or mononuclear cells (MNC) do not expand well. Such cell selection processes often result in low CD34+ cell recoveries and suboptimal purities, with a consequent lowering of total overall cell expansion. CD52 is an abundant antigen specifically expressed on lymphocytes, monocytes, eosinophils, thymocytes and macrophages; and there is significant evidence for the lack of CD52 expression on hematopoietic progenitor cells. In our current study, Campath-1H, a humanized anti-CD52 antibody, was added to Ficoll-separated cord blood cells to promote active selection of hematopoietic stem cells and curtailment of excess lymphocyte expansion in ex vivo cultures. Prior to start of the cultures, initial Ficoll separation of cord blood removed most of the granulocytes (CD33+ cells: 2.3%, CD13+ cells: 3.1%) though 90.3% percent of the cells remained CD52 positive. Megakaryocytic (CD41+), erythroid (GlyA+) and CD34+ cell percentages in the starting cultures were 12.8%, 1.8% and 0.4% respectively. Cells were not subjected to CD34 or CD133 column selection prior to ex vivo expansion and Campath-1H was added every 3 days to the expansion culture media (incorporating 50 ng/ml SCF, TPO and Flt-3 ligand in StemSpan SFEM medium). CD52+ cells decreased from 90.3% to 3.1% after treatment with Campath-1H antibody (Ab), maintaining at 64.4% in control cultures. After 2 weeks of culture, CD34 cells expanded 12.56 fold in cultures with Campath-1H added versus 7.10 fold in cultures without Campath. There was a 1.49 fold decrease in total cell number in ex-vivo cultures without Campath-1H added, which was consistent with the current literature on expansion with non-selected cells. With Campath-1H in the culture system, there was a 1.60 fold increase in total cell number, which was 2.39 fold higher than control cultures. Decrease in the lymphoid cell percentage was accompanied by a relative increase in CD33+ (50.2% with Ab vs. 41.9% without Ab) and CD13+ (82% with Ab vs. 61.1% without Ab) myeloid cells, CD41+ megakaryocytes (34.5% with Ab vs 13.0% without Ab) and GlyA+ erythrocytes (9.9% with Ab vs. 4.5% without Ab). The relative expansion of non-lymphocyte populations observed in our cultures system could potentially reduce graft-versus-host disease (GVHD) and promote enhanced engraftment of myeloid, megakaryocytic and erythroid precursors after transplantation. The ability to use a CD34/CD133 column-independent expansion system through Ab-regulated culture with a clinically licensed antibody is also compelling.

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Synergism between erythropoietin and interleukin-3 in the induction of hematopoietic stem cell proliferation and erythroid burst colony formation

Blood ◽

10.1182/blood.v72.3.944.944 ◽

1988 ◽

Vol 72 (3) ◽

pp. 944-951 ◽

Cited By ~ 2

Author(s):

G Migliaccio ◽

AR Migliaccio ◽

JW Visser

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Cell Number ◽

Total Cell ◽

Recombinant Erythropoietin ◽

Erythroid Progenitors ◽

Total Cell Number ◽

Hematopoietic Stem ◽

Interleukin 3 ◽

Stem And Progenitor Cells

Abstract The influence of recombinant erythropoietin (Ep) and interleukin-3 (IL- 3) on the proliferation and differentiation of murine hematopoietic stem and progenitor cells was investigated in serum-deprived cultures. The differentiation of progenitor cells, purified by collecting blast cell colonies from spleen cell cultures of 5-fluorouracil-treated mice, was evaluated by scoring the number and type of colonies appearing after eight days in semisolid culture. IL-3 induced the formation of both erythroid and granulocyte-macrophage colonies in a concentration- dependent fashion, the plateau being reached at 300 U/mL. However, concentrations of IL-3 alone that had little or no effect (less than or equal to 10 U/mL) induced maximal numbers of erythroid bursts in the presence of Ep (1.5 IU/mL). In the presence of Ep alone, no colonies were seen. Proliferation of quiescent hematopoietic stem cells, purified by cell sorting and evaluated by spleen colony assay (CFU-S), was investigated by measuring the total cell number and CFU-S content and the DNA histogram at 20 and 48 hours of liquid culture. Almost no cells or CFU-S survived 20 hours of incubation without the addition of IL-3. The presence of either IL-3 (400 U/mL) or the combination of EP and IL-3 (10 U/mL), supported the maintenance of nearly 40% of sorted CFU-S for 48 hours. Approximately 10% of these cells were in the S phase of the cell cycle at 20 hours and an increase in the total cell number per culture, but not in the CFU-S content, was detected at 48 hours. These data indicate that IL-3 exerts a differentiative and proliferative effect on early stem and progenitor cells, which is concentration dependent. At IL-3 concentrations, which had little or no activity alone, Ep acted synergistically to induce both proliferation of stem cells and differentiation of erythroid progenitors.

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Synergism between erythropoietin and interleukin-3 in the induction of hematopoietic stem cell proliferation and erythroid burst colony formation

Blood ◽

10.1182/blood.v72.3.944.bloodjournal723944 ◽

1988 ◽

Vol 72 (3) ◽

pp. 944-951 ◽

Cited By ~ 51

Author(s):

G Migliaccio ◽

AR Migliaccio ◽

JW Visser

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Cell Number ◽

Total Cell ◽

Recombinant Erythropoietin ◽

Erythroid Progenitors ◽

Total Cell Number ◽

Hematopoietic Stem ◽

Interleukin 3 ◽

Stem And Progenitor Cells

The influence of recombinant erythropoietin (Ep) and interleukin-3 (IL- 3) on the proliferation and differentiation of murine hematopoietic stem and progenitor cells was investigated in serum-deprived cultures. The differentiation of progenitor cells, purified by collecting blast cell colonies from spleen cell cultures of 5-fluorouracil-treated mice, was evaluated by scoring the number and type of colonies appearing after eight days in semisolid culture. IL-3 induced the formation of both erythroid and granulocyte-macrophage colonies in a concentration- dependent fashion, the plateau being reached at 300 U/mL. However, concentrations of IL-3 alone that had little or no effect (less than or equal to 10 U/mL) induced maximal numbers of erythroid bursts in the presence of Ep (1.5 IU/mL). In the presence of Ep alone, no colonies were seen. Proliferation of quiescent hematopoietic stem cells, purified by cell sorting and evaluated by spleen colony assay (CFU-S), was investigated by measuring the total cell number and CFU-S content and the DNA histogram at 20 and 48 hours of liquid culture. Almost no cells or CFU-S survived 20 hours of incubation without the addition of IL-3. The presence of either IL-3 (400 U/mL) or the combination of EP and IL-3 (10 U/mL), supported the maintenance of nearly 40% of sorted CFU-S for 48 hours. Approximately 10% of these cells were in the S phase of the cell cycle at 20 hours and an increase in the total cell number per culture, but not in the CFU-S content, was detected at 48 hours. These data indicate that IL-3 exerts a differentiative and proliferative effect on early stem and progenitor cells, which is concentration dependent. At IL-3 concentrations, which had little or no activity alone, Ep acted synergistically to induce both proliferation of stem cells and differentiation of erythroid progenitors.

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The structure of pre-mRNA and mRNA from erythroid cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082030 ◽

1978 ◽

Vol 36 (2) ◽

pp. 234-235

Author(s):

A.-M. Ladhoff ◽

B.J. Thiele ◽

Ch. Coutelle ◽

S. Rosenthal

Keyword(s):

Bone Marrow ◽

Structural Transformation ◽

Electron Micrographs ◽

Ammonium Acetate ◽

Bone Marrow Cells ◽

Erythroid Cells ◽

Globin Mrna ◽

Ordered Structures ◽

Rabbit Bone ◽

Rabbit Reticulocytes

The suggested precursor-product relationship between the nuclear pre-mRNA and the cytoplasmic mRNA has created increased interest also in the structure of these RNA species. Previously we have been published electron micrographs of individual pre-mRNA molecules from erythroid cells. An intersting observation was the appearance of a contour, probably corresponding to higher ordered structures, on one end of 10 % of the pre-mRNA molecules from erythroid rabbit bone marrow cells (Fig. 1A). A virtual similar contour was observed in molecules of 9S globin mRNA from rabbit reticulocytes (Fig. 1B). A structural transformation in a linear contour occurs if the RNA is heated for 10 min to 90°C in the presence of 80 % formamide. This structural transformation is reversible when the denatured RNA is precipitated and redissolved in 0.2 M ammonium acetate.

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Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽

10.1017/s0967199416000228 ◽

2016 ◽

Vol 24 (6) ◽

pp. 890-899 ◽

Cited By ~ 4

Author(s):

A.L.S. Guimarães ◽

S.A. Pereira ◽

M. N. Diógenes ◽

M.A.N. Dode

Keyword(s):

Ascorbic Acid ◽

Cell Number ◽

Total Cell ◽

Control Group ◽

Bovine Embryo ◽

Total Cell Number ◽

Embryo Production ◽

Embryo Size ◽

Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

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The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽

10.1242/dev.102.4.793 ◽

1988 ◽

Vol 102 (4) ◽

pp. 793-803 ◽

Cited By ~ 3

Author(s):

V.E. Papaioannou ◽

K.M. Ebert

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Staining Technique ◽

Cell Number ◽

Total Cell ◽

Differential Staining ◽

Morphological Development ◽

Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

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Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽

10.1017/s0967199408005005 ◽

2009 ◽

Vol 17 (1) ◽

pp. 57-61 ◽

Cited By ~ 4

Author(s):

M. Popelková ◽

Z. Turanová ◽

L. Koprdová ◽

A. Ostró ◽

S. Toporcerová ◽

...

Keyword(s):

Cell Number ◽

Total Cell ◽

Control Group ◽

Assisted Hatching ◽

Hatching Rate ◽

Total Cell Number ◽

Developmental Potential ◽

Significant Difference ◽

Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

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Effect of follicular fluid supplementation during in vitro maturation on total cell number in bovine blastocysts produced in vitro

Revista Brasileira de Zootecnia ◽

10.1590/s1516-35982014000300003 ◽

2014 ◽

Vol 43 (3) ◽

pp. 120-126 ◽

Cited By ~ 5

Author(s):

Maria Helena Coelho Cruz ◽

Naiara Zoccal Saraiva ◽

Jurandir Ferreira da Cruz ◽

Clara Slade Oliveira ◽

Maite Del Collado ◽

...

Keyword(s):

Follicular Fluid ◽

In Vitro Maturation ◽

Cell Number ◽

Total Cell ◽

Total Cell Number ◽

Fluid Supplementation

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Hematopoietic stem and progenitor cells are present in healthy gingiva tissue

Journal of Experimental Medicine ◽

10.1084/jem.20200737 ◽

2021 ◽

Vol 218 (4) ◽

Author(s):

Siddharth Krishnan ◽

Kelly Wemyss ◽

Ian E. Prise ◽

Flora A. McClure ◽

Conor O’Boyle ◽

...

Keyword(s):

Bone Marrow ◽

Immune Responses ◽

Progenitor Cells ◽

Myeloid Cell ◽

Extramedullary Hematopoiesis ◽

Innate Immune ◽

Innate Immune Cells ◽

Hematopoietic Stem ◽

Effector Cells ◽

Stem And Progenitor Cells

Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier.

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Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos

Zygote ◽

10.1017/s0967199400003890 ◽

1997 ◽

Vol 5 (4) ◽

pp. 309-320 ◽

Cited By ~ 49

Author(s):

Rabindranath de la Fuente ◽

W. Allan King

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Total Cell ◽

Differential Staining ◽

Similar Proportion ◽

Bovine Embryos ◽

Total Cell Number ◽

Cell Numbers

SummaryThe mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

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