Bmi-1 Regulates Ex Vivo Self-Renewal of Human Erythroblasts

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 844-844
Author(s):  
Jayme L. Olsen ◽  
Judith Aeschlimann ◽  
Connie M. Westhoff ◽  
James Palis
Keyword(s):  
Ex Vivo ◽  
Human Peripheral Blood ◽  
The United States ◽  
Blood Group Antigens ◽  
Proliferative Capacity ◽  
Transfusion Therapy ◽  
Self Renewal ◽  
Erythroid Precursors ◽  
Necessary And Sufficient

Abstract About twelve million units of red blood cells (RBCs) are transfused yearly in the United States. Ex vivo cultured RBCs could serve as a supplemental source to treat alloimmunized patients requiring chronic transfusions. The limited proliferative capacity of erythroid precursors is a major obstacle to generating sufficient numbers of RBCs to constitute even one unit of blood. We previously determined that erythroblasts derived from the murine embryo have a unique ability to self-renew extensively ex vivo, and that Bmi-1, a member of the polycomb repressive complex 1 (PRC1), is both necessary and sufficient to drive the extensive self-renewal of murine bone marrow-derived erythroblasts. Here, we tested the hypothesis that Bmi-1 regulates the ex vivo self-renewal of human erythroid cells. Bmi-1 overexpression increased the proliferative capacity of adult human peripheral blood mononuclear cell-derived erythroblasts, which normally have restricted ex vivo self-renewal, more than 10 billion-fold. Bmi-1-induced self-renewing erythroblasts (iSREs) retained an immature erythroid morphology and cell surface phenotype throughout culture. Chemical inhibition of Bmi-1 led to collapse of the culture, with a massive reduction in cycling cells and an increase in apoptosis. Taken together, these data indicate that Bmi-1 in iSREs is both necessary and sufficient to increase the self-renewal capacity of human erythroid precursors. Importantly, Bmi-1 overexpression does not interfere with the ability of the iSREs to mature into reticulocytes in vitro. Serological analysis of multiple blood group antigens demonstrated that iSRE-derived reticulocytes express the same antigens as the donor's RBCs. Bmi-1 can act as part of canonical or non-canonical PRC1 and different complex members have been associated with hematopoietic self-renewal versus differentiation. Expression studies of self-renewing human erythroblasts revealed that the non-canonical PRC1 member, RYBP, was expressed at higher levels than canonical PRC1 members. In addition, maturation of erythroid precursors is associated with down-regulation of Bmi-1 and non-canonical PRC1 members, as well as the upregulation of several canonical members. Knockdown of RYBP reduced the proliferative capacity of the iSREs, suggesting that Bmi-1 regulates erythroid self-renewal through non-canonical PRC1 interactions. Consistent with this hypothesis, preliminary studies inhibiting canonical PRC1 members led to an additional several trillion-fold increase in the ex vivo proliferative capacity of human iSREs compared to vehicle-treated control cultures. These data suggest that Bmi-1 regulates erythroid self-renewal through non-canonical PRC1. Increasing the ex vivo self-renewal capacity of human erythroblasts ultimately paves the way for the generation of sufficient numbers of cultured RBCs for blood typing and transfusion therapy, as well as the establishment of in vitro models of RBC-intrinsic disorders. Disclosures Palis: Rubies Therapeutics: Consultancy.

scholarly journals Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus

Blood ◽  
2011 ◽  
Vol 117 (9) ◽  
pp. 2708-2717 ◽  
Author(s):  
Samantha J. England ◽  
Kathleen E. McGrath ◽  
Jenna M. Frame ◽  
James Palis
Keyword(s):  
Stem Cell ◽  
Glucocorticoid Receptors ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Erythroid Progenitors ◽  
Transfusion Therapy ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Erythroid Precursors ◽  
Definitive Erythropoiesis

Abstract In the hematopoietic hierarchy, only stem cells are thought to be capable of long-term self-renewal. Erythroid progenitors derived from fetal or adult mammalian hematopoietic tissues are capable of short-term, or restricted (102- to 105-fold), ex vivo expansion in the presence of erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary erythroid precursors derived from early mouse embryos are capable of extensive (106- to 1060-fold) ex vivo proliferation. These cells morphologically, immunophenotypically, and functionally resemble proerythroblasts, maintaining both cytokine dependence and the potential, despite prolonged culture, to generate enucleated erythrocytes after 3-4 maturational cell divisions. This capacity for extensive erythroblast self-renewal is temporally associated with the emergence of definitive erythropoiesis in the yolk sac and its transition to the fetal liver. In contrast, hematopoietic stem cell-derived definitive erythropoiesis in the adult is associated almost exclusively with restricted ex vivo self-renewal. Primary primitive erythroid precursors, which lack significant expression of Kit and glucocorticoid receptors, lack ex vivo self-renewal capacity. Extensively self-renewing erythroblasts, despite their near complete maturity within the hematopoietic hierarchy, may ultimately serve as a renewable source of red cells for transfusion therapy.

Repurposing N,N-Dimethylacetamide (DMA), a Pharmaceutical Excipient, as a Prototype Novel Anti-inflammatory Agent for the Prevention and/or Treatment of Preterm Birth

2018 ◽  
Vol 24 (9) ◽  
pp. 989-992 ◽  
Author(s):  
Samir Gorasiya ◽  
Juliet Mushi ◽  
Ryan Pekson ◽  
Sabesan Yoganathan ◽  
Sandra E. Reznik
Keyword(s):  
Preterm Birth ◽  
Ex Vivo ◽  
The United States ◽  
Occurrence Rate ◽  
Pharmaceutical Excipient ◽  
Ongoing Work ◽  
Exciting Line ◽  
Pregnant Mice

Background: Preterm birth (PTB), or birth that occurs before 37 weeks of gestation, accounts for the majority of perinatal morbidity and mortality. As of 2016, PTB has an occurrence rate of 9.6% in the United States and accounts for up to 18 percent of births worldwide. Inflammation has been identified as the most common cause of PTB, but effective pharmacotherapy has yet to be developed to prevent inflammation driven PTB. Our group has discovered that N,N-dimethylacetamide (DMA), a readily available solvent commonly used as a pharmaceutical excipient, rescues lipopolysaccharide (LPS)-induced timed pregnant mice from PTB. Methods: We have used in vivo, ex vivo and in vitro approaches to investigate this compound further. Results: Interestingly, we found that DMA suppresses cytokine secretion by inhibiting nuclear factor-kappa B (NF-κB). In ongoing work in this exciting line of investigation, we are currently investigating structural analogs of DMA, some of them novel, to optimize this approach focused on the inflammation associated with PTB. Conclusion: Successful development of pharmacotherapy for the prevention of PTB rests upon the pursuit of multiple strategies to solve this important clinical challenge.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Icodextrin Effluent Leads to a Greater Proliferation than Glucose Effluent of Human Mesothelial Cells Studied Ex Vivo

2000 ◽  
Vol 20 (6) ◽  
pp. 742-747 ◽  
Author(s):  
M.-Auxiliadora Bajo ◽  
Rafael Selgas ◽  
M.-Angeles Castro ◽  
Gloria Del Peso ◽  
Cándido Díaz ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Tertiary Care ◽  
Mesothelial Cell ◽  
Public University ◽  
Mesothelial Cells ◽  
University Hospital ◽  
Proliferative Capacity ◽  
Initial Culture ◽  
Effect Of Glucose

Objective To compare the effect of glucose (Glu) and icodextrin (Ico) dialysate on in vitro culture of mesothelial cells (MC) from peritoneal dialysis (PD) patients. Design Prospective, controlled comparative study on the effects of two PD solutions. Setting A tertiary-care public university hospital. Patients Sixteen PD patients regularly using Glu dialysate were asked to collect an 8-hour dwell peritoneal effluent on 2 different days, with an interval shorter than 7 days. In the first collection, 2.27% Glu solution and in the last, 7.5% Ico solution was infused. Human MC were isolated from the nocturnal peritoneal effluent bags and grown ex vivo. Main Outcome Measures Mesothelial cell proliferative capacity ex vivo. Results Mesothelial cells were present in all patient dialysates except that of a single patient's Glu dialysate. The number of MC drained was similar with both solutions. After the initial culture reached confluence, MC were identified in 14 and 12 patients receiving Ico and Glu, respectively. However, in 1 patient using Ico and in 2 using Glu, the MC count at this stage was so low that further subculture could not be performed. Cells from Ico-derived solutions exhibited a higher degree of proliferation than cells from Glu-derived solutions. The morphology of MC was also different. Cells from drained effluent were typical in 11 patients using Glu solution in contrast with 14 patients using Ico. At confluence, the percentages of typical appearance were 50% and 92.9% ( p < 0.05) in Glu and Ico respectively. Conclusions Mesothelial cells taken from icodextrin effluent show a greater proliferation ex vivo than those taken from glucose effluent.

scholarly journals A novel O -linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

Open Biology ◽  
2014 ◽  
Vol 4 (1) ◽  
pp. 130202 ◽  
Author(s):  
Jafar Mahdavi ◽  
Necmettin Pirinccioglu ◽  
Neil J. Oldfield ◽  
Elisabet Carlsohn ◽  
Jeroen Stoof ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Blood Group ◽  
Campylobacter Jejuni ◽  
Outer Membrane Protein ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Major Outer Membrane Protein ◽  
Blood Group Antigens

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168 . Significantly, the MOMP was shown to be O- glycosylated at Thr 268 ; previously only flagellin proteins were known to be O- glycosylated in C. jejuni . Substitution of MOMP Thr 268 led to significantly reduced binding to BgAgs. The O- glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr 268 ; modelling suggested that O- glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr 268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni , confirming the significance of this O- glycosylation in pathogenesis.

Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element

Blood ◽  
2003 ◽  
Vol 102 (13) ◽  
pp. 4369-4376 ◽  
Author(s):  
James C. Mulloy ◽  
Jorg Cammenga ◽  
Francisco J. Berguido ◽  
Kaida Wu ◽  
Ping Zhou ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Cells ◽  
The Self ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Cd34

AbstractHematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

scholarly journals Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 545-556 ◽  
Author(s):  
JC Young ◽  
A Varma ◽  
D DiGiusto ◽  
MP Backer
Keyword(s):  
Cell Culture ◽  
Single Cell ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Proliferative Capacity ◽  
Quiescent Cells ◽  
Cell Divisions ◽  
Single Cell Culture ◽  
Bulk Culture

Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells. Ex vivo expansion of this population is proposed as a means of providing accelerated short-term, as well as long-term, engraftment after myeloablative therapy. Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibiting quiescent behavior during culture. CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells on a murine stromal layer in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking of the number of cell divisions. Progenitor cells present after a 2-week expansion period were sorted into CD34+/Lin-/dyebright and CD34+/Lin- /dyedim fractions and then cultured in a 4-week single-cell proliferation assay to characterize the proliferative capacity of each group. Fifty-nine percent of progenitors remaining dyebright after bulk culture (four or fewer cell divisions) were observed to proliferate in single cell culture, and produced an average of 1,780 cells per plated cell. In contrast, only 26% of dyedim (more than four divisions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell. Similar behaviors were observed after a second consecutive cycle of bulk culture, indicating that quiescent cells with high proliferative capacity existed in culture for at least 4 weeks. Single CD34+/Lin-/dyebright progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and these progeny included multilineage colony forming cells. These data demonstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent behavior. The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1686-1686
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture ◽  
Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

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