An RNA Interference Model of RPS19 Deficiency in Diamond Blackfan Anemia Recapitulates Defective Erythropoiesis and Rescue by Dexamethasone: Identification of Dexamethasone Responsive Genes by Microarray.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 721-721 ◽  
Author(s):  
Benjamin L. Ebert ◽  
Michele Lee ◽  
Jennifer Pretz ◽  
Todd R. Golub ◽  
Colin Sieff
Keyword(s):  
Progenitor Cells ◽  
Molecular Basis ◽  
Cellular Proliferation ◽  
Erythroid Differentiation ◽  
Regulatory Genes ◽  
Cell Surface Expression ◽  
Erythroid Progenitor ◽  
Diamond Blackfan Anemia ◽  
Erythroid Progenitor Cells ◽  
Cd34 Cells

Abstract Diamond Blackfan Anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation, but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but the relevance of this ubiquitously expressed ribosomal gene to erythroid differentiation is unclear. Glucocorticoids are the primary pharmacological therapy for patients with DBA, but the molecular basis for the activity of glucocorticoids in erythroid differentiation has not been identified. Through retroviral expression of short hairpin RNAs (shRNAs), we demonstrate that targeted degradation of the RPS19 gene in cultured human CD34+ cells blocks the proliferation and differentiation of erythroid progenitor cells. Decreased RPS19 expression limited production of erythroid colony formation on methylcellulose, decreased cell surface expression of glycophorin-A, and decreased cellular proliferation. Treatment of RPS19 deficient cells with dexamethasone restored erythroid differentiation to normal levels. We investigated the molecular basis of pharmacologic therapies for DBA using oligonucleotide microarrays to survey gene expression in CD34+ cells treated with combinations of erythropoietin, dexamethasone, IL-3, and SCF. None of these agents had a direct effect on the expression of RPS19 or on the coordinate expression of other ribosomal genes. Instead, dexamethasone activated a genetic program that includes a set of key hematopoietic regulatory genes, including Flt-3 and PLZF, that increase proliferation of hematopoietic progenitor cells. Genes specific to erythroid progenitor cells were up-regulated by dexamethasone, while genes specific to non-erythroid lineages were powerfully down-regulated. Deficiency of RPS19 therefore blocks proliferation of immature erythroid progenitor cells, and dexamethasone activates proliferation of the same cell population through mechanisms independent of RPS19. Identification of the key regulatory genes affected by dexamethasone may aid in the development of novel therapeutics that de-couple the beneficial hematopoietic effects of dexamethasone from detrimental non-hematopoietic side effects.

scholarly journals An RNA interference model of RPS19 deficiency in Diamond-Blackfan anemia recapitulates defective hematopoiesis and rescue by dexamethasone: identification of dexamethasone-responsive genes by microarray

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4620-4626 ◽  
Author(s):  
Benjamin L. Ebert ◽  
Michele M. Lee ◽  
Jennifer L. Pretz ◽  
Aravind Subramanian ◽  
Raymond Mak ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Molecular Basis ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Diamond Blackfan Anemia ◽  
Human Cd34 ◽  
Erythroid Progenitor Cells ◽  
Cd34 Cells ◽  
Stimulation Of Erythropoiesis ◽  
Short Hairpin Rnas

AbstractDiamond-Blackfan anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but its relevance to erythroid differentiation is unclear. The molecular basis for the stimulation of erythropoiesis by glucocorticoids in patients with DBA has not been identified. We demonstrate that targeted degradation of the RPS19 transcript, through retroviral expression of short hairpin RNAs (shRNAs), blocks the proliferation and differentiation of erythroid progenitor cells in cultured human CD34+ cells. Treatment of RPS19-deficient cells with dexamethasone restores erythroid differentiation to normal levels. We investigated the molecular basis of pharmacologic therapies for DBA using oligonucleotide microarrays to survey gene expression in CD34+ cells treated with combinations of dexamethasone, erythropoietin, stem cell factor, and interleukin-3. Dexamethasone did not alter expression of RPS19 but activated a genetic program that includes a set of key hematopoietic regulatory genes. Genes specific to erythroid progenitor cells were up-regulated by dexamethasone, while genes specific to nonerythroid lineages were down-regulated. Deficiency of RPS19 therefore blocks proliferation of immature erythroid progenitor cells, and dexamethasone activates proliferation of the same cell population through mechanisms independent of RPS19. (Blood. 2005;105:4620-4626)

scholarly journals Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1576-1582 ◽  
Author(s):  
M Silva ◽  
D Grillot ◽  
A Benito ◽  
C Richard ◽  
G Nunez ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Apoptotic Cell ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Survival Factor ◽  
Erythroid Progenitor Cells ◽  
Survival Signal ◽  
Proliferation And Differentiation

Abstract Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.

scholarly journals Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 572-580 ◽  
Author(s):  
K Muta ◽  
SB Krantz ◽  
MC Bondurant ◽  
CH Dai
Keyword(s):  
Stem Cell ◽  
Cell Viability ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Single Cells ◽  
Erythroid Differentiation ◽  
Tyrosine Kinase Receptor ◽  
Total Production ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

Stem cell factor (SCF), the ligand for the c-kit tyrosine kinase receptor, markedly stimulates the accumulation of erythroid progenitor cells in vitro. We now report that SCF delays erythroid differentiation among the progeny of individual erythroid progenitors while greatly increasing the proliferation of these progeny. These effects appear to be independent of an effect on maintenance of cell viability. Highly purified day-6 erythroid colony-forming cells (ECFC), consisting mainly of colony-forming units-erythroid (CFU-E), were generated from human peripheral blood burst-forming units-erythroid (BFU-E). Addition of SCF to the ECFC in serum-free liquid culture, together with erythropoietin (EP) and insulin-like growth factor 1 (IGF-1), resulted in a marked increase in DNA synthesis, associated with a delayed peak in cellular benzidine positivity and a delayed incorporation of 59Fe into hemoglobin compared with cultures without SCF. In the presence of SCF, the number of ECFC was greatly expanded during this culture period, and total production of benzidine-positive cells plus hemoglobin synthesis were ultimately increased. To determine the effect of SCF on individual ECFC, single-cell cultures were performed in both semisolid and liquid media. These cultures demonstrated that SCF, in the presence of EP and IGF-1, acted on single cells and their descendants to delay erythroid differentiation while substantially stimulating cellular proliferation, without an enhancement of viability of the initial cells. This was also evident when the effect of SCF was determined using clones of ECFC derived from single BFU-E. Our experiments demonstrate that SCF acts on individual day-6 ECFC to retard erythroid differentiation while simultaneously providing enhanced proliferation by a process apparently independent of an effect on cell viability or programmed cell death.

Chromatin Modifying Agents Promote the Ex Vivo Production of Functional Human Erythroid Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 340-340
Author(s):  
Pratima Chaurasia ◽  
Dmitriy Berenzon ◽  
Ronald Hoffman
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylase Inhibitors ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Significant Proportion ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Cell Product ◽  
Erythroid Progenitor Cells ◽  
Cd34 Cells

Abstract Abstract 340 Presently, blood transfusion products (TP) are composed of terminally differentiated cells with a finite life span. We attempted to develop an alternative TP which would be capable of generating additional red blood cells (RBC). Several histone deacetylase inhibitors (HDACIs) were used in vitro to reprogram cord blood (CB) CD34+ cells to differentiate to erythroid progenitor cells (EPC). We demonstrated that CB CD34+ cells in the presence of HDACIs (SAHA, VPA and TSA), and a combination of cytokines SCF, IL-3, TPO and FLT3, promoted expansion of CD34+ cells and CD34+CD90+ cells as compared to cultures containing cytokines alone. Addition of VPA resulted in the greatest expansion of CD34+ cells, CD34+CD90+cells+ (59.4 fold, p=0.01; 66.7 fold, p=0.02, respectively) as compared to SAHA and TSA. VPA also led to the generation of the greatest absolute number of EPC cells (14.9×106, p=0.002), approximately a 5500 fold in the numbers of assayable EPC, as compared to primary CB. The single cell analyses of CB CD34+ cells (Day0) and single CD34+ reisolated from ex-vivo cultures pretreated with cytokines alone or cytokines+VPA demonstrated an skewed differentiation program of CD34+ cells to EPC (>94%, p=0.003) compared to CB CD34+(50%) and cytokines alone (29%). We investigated the expression of lineage specific phenotypic markers expressed by CD34+ cells exposed to cytokines alone or cytokines plus VPA. The FACS analyses showed a significantly greater proportion of CD34+CD36+ (52.4% vs 21.0%) CD36+CD71+(44.5% vs7.6%), CD36+GPA+(12.8% Vs 4.0%) and CD71+GPA+(22.2% vs 6.3%) cells with lower numbers of CD19+(2.8% vs 13.6%) cells, CD14+(2.0% vs 8.9%), CD15+(1.8 vs 6.9%) in VPA treated CD34+ cells as compared to cytokines alone. We monitored the relative expression of a group of genes characteristic of both primitive HPC and erythroid commitment (Bmi1, Dnmt1, Ezh2, Smad5, Eklf, GATA1, GATA2, EpoR and Pu.1). Q-PCR was performed on CD34+cells reisolated from cultures treated with cytokines alone or cytokines plus VPA and compared to primary CB CD34+ cells. The expression of genes associated with retention of the biological properties of the primitive HPC (Bmi1-2.6 fold, Dnmt1-10.3 fold and Ezh2-4.8 fold) and erythroid lineage specific genes (Smad5-6.2 fold, GATA2-3.7 fold) were upregulated and Pu.1 (0.6-fold), GATA1(1.9 fold) were downregulated as compared to cytokines alone. However, expression of EpoR and Eklf were similar in the two cell populations Histone acetylation study showed that the CB CD34+ cells and VPA treated CD34+ cells had a significant proportion of acetylated H3K9 cells, 52.2% and 56.1% respectively, while this population was virtually absent in CD34+ cells exposed to cytokines alone (1.3%, p=0.001). ChIP assay demonstrated a varying degree of H3K9/14 and H3K27 acetylation within the promoters of VPA treated CD34+ cells for GATA2 (7.4 fold, 7.2 fold), Eklf (7.4 fold, 9.7 fold), Pu.1(4.5fold, 4.8 fold), EpoR (2.3 fold, 4.7 fold) and GATA1(4.7 fold, 2.9 fold). The acetylation of cytokines treated CD34+ cells were much lower than VPA treated CD34+ cells. The VPA treated cell product after 9 days (supplemented with SCF, Epo and IL-3 for 2 additional days) compared to 7 days contained a greater percentage of EPC and erythroid precursor cells CD34+CD36+(24.9% vs 23.0%), CD36+GPA+(33.9% vs 18.8%), CD36+. CD71+(55.8% vs 37.8%), CD71+GPA+(33.9% vs 20.5%) and CD34+CXCR4+(28.8% vs 21.0 %). The TP contained very limited number of CD19+(1.4%), CD14+(11.11%) or CD15+(6.8%) of cells. Approximately 50 % of the cells present in the TP expressed the chemokine receptor CXCR4. We next evaluated the behavior of ex vivo expanded cell product following transfusion into sublethally irradiated NOD/SCID mice. FACS analyses of mice peripheral blood (PB) on serial days showed evidence of circulating nucleated erythroid and enucleated red cells. The greatest number of circulating human RBC (12.4%±6.8%) was observed on day5. RT-PCR analyses on the PB of mice on day 15 revealed the presence of erythroid cells containing both human adult and fetal hemoglobin. On day 15 the mice were sacrificed and the degree of human cells engraftment in the marrow were predominately hu -CD45+ (7.4%), CD34-CD36+(1.8%), CD36 (4.5%) and GPA+(1.7%) with no evidence of CD33+, CD14+, CD19+ and CD41+ cells. The ex vivo generated EPC-TP likely represents a paradigm shift in transfusion medicine due to its continued ability to generate additional RBC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutant N-RAS Induces Erythroid Lineage Dysplasia in Human CD34+ Cells

1997 ◽  
Vol 185 (7) ◽  
pp. 1337-1348 ◽  
Author(s):  
Richard L. Darley ◽  
Terence G. Hoy ◽  
Paul Baines ◽  
Rose Ann Padua ◽  
Alan K. Burnett
Keyword(s):  
Myelodysplastic Syndrome ◽  
Progenitor Cells ◽  
Marker Gene ◽  
Erythroid Differentiation ◽  
Multiparameter Flow Cytometry ◽  
Lacz Gene ◽  
Erythroid Progenitor ◽  
Stage Of Development ◽  
Cd34 Cells ◽  
Mutational Activation

RAS mutations arise at high frequency (20–40%) in both acute myeloid leukemia and myelodysplastic syndrome (which is considered to be a manifestation of preleukemic disease). In each case, mutations arise predominantly at the N-RAS locus. These observations suggest a fundamental role for this oncogene in leukemogenesis. However, despite its obvious significance, little is known of how this key oncogene may subvert the process of hematopoiesis in human cells. Using CD34+ progenitor cells, we have modeled the preleukemic state by infecting these cells with amphotropic retrovirus expressing mutant N-RAS together with the selectable marker gene lacZ. Expression of the lacZ gene product, β-galactosidase, allows direct identification and study of N-RAS–expressing cells by incubating infected cultures with a fluorogenic substrate for β-galactosidase, which gives rise to a fluorescent signal within the infected cells. By using multiparameter flow cytometry, we have studied the ability of CD34+ cells expressing mutant N-RAS to undergo erythroid differentiation induced by erythropoietin. By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle. This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development. The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program. This erythroid lineage dysplasia recapitulates one of the most common features of myelodysplastic syndrome, and for the first time provides a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia.

scholarly journals Inhibition of Immature Erythroid Progenitor Cell Proliferation by Macrophage Inflammatory Protein-1α by Interacting Mainly With a C-C Chemokine Receptor, CCR1

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 605-611 ◽  
Author(s):  
Shao-bo Su ◽  
Naofumi Mukaida ◽  
Jian-bin Wang ◽  
Yi Zhang ◽  
Akiyoshi Takami ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Inflammatory Protein ◽  
Cd34 Cells ◽  
Macrophage Inflammatory Protein ◽  
Marrow Cells

Abstract Several lines of evidence indicate that macrophage inflammatory protein-1α (MIP-1α) modulates the proliferation of hematopoietic progenitor cells, depending on their maturational stages. To clarify the mechanisms for the modulation of hematopoiesis by this chemokine, we examined the expression of a receptor for MIP-1α, CCR1, on bone marrow cells of normal individuals using a specific antibody and explored the effects of MIP-1α on in vitro erythropoiesis driven by stem cell factor (SCF) and erythropoietin (Epo). CCR1 was expressed on glycophorin A-positive erythroblasts in addition to lymphocytes and granulocytes. CCR1+ cells, isolated from bone marrow mononuclear cells (BMMNCs) using a cell sorter, comprised virtually all erythroid progenitor cells in the BMMNCs. Moreover, MIP-1α inhibited, in a dose-dependent manner, colony formation by burst-forming unit-erythroid (BFU-E), but not by colony forming unit-erythroid (CFU-E), in a methylcellulose culture of purified human CD34+ bone marrow cells. Although reverse-transcription polymerase chain reaction (RT-PCR) showed the presence of CCR1, CCR4, and CCR5 transcripts in CD34+ cells in BM, anti-CCR1 antibodies significantly abrogated the inhibitory effects of MIP-1α on BFU-E formation both in a methylcellulose culture and in a single cell proliferation assay of purified CD34+ cells. Although the contribution of CCR4 or CCR5 cannot be completely excluded, these results suggest that MIP-1α–mediated suppression of the proliferation of immature, but not mature erythroid progenitor cells, is largely mediated by CCR1 expressed on these progenitor cells.

Lineage-Specific Activation of p53 in Response to Ribosomal Haploinsufficiency in Human Bone Marrow Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 948-948
Author(s):  
Shilpee Dutt ◽  
Anupama Narla ◽  
Jeffery Lorne Kutok ◽  
Benjamin L. Ebert
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Intense Staining ◽  
Bone Marrow Biopsies ◽  
Erythroid Progenitor Cells ◽  
Cd34 Cells

Abstract Abstract 948 Haploinsufficiency for the ribosomal protein genes RPS14 and RPS19 have been implicated in the erythroid defect in the 5q- syndrome and Diamond Blackfan Anemia, respectively. However, the mechanism by which defective ribosome biogenesis causes erythroid failure is unknown. In this study, we found that shRNA mediated knockdown of RPS14 or RPS19 in primary human CD34+ cells stabilize TP53 by day 4 after infection with concomitant arrest of these cells at G1 stage of cell cycle. The levels of TP53 attained are comparable to the levels observed following gamma irradiation (5Gy) of the CD34+ cells. Using quantitative PCR, we confirmed that stabilized TP53 activates expression of downstream target genes MDM2, p21, Bax and Wig-1. Furthermore, treatment of the CD34+ cells with Nutlin-3 phenocopies RPS14 or RPS19 knockdown, suggesting that the mechanism of TP53 activation is mediated by MDM2 pathway. Conversely, treatment with pifithrin-alpha, which inhibits the transactivation activity of TP53, rescues the effects of RPS14 or RPS19 knockdown. The in vitro activation of TP53 in CD34+ cells was restricted to erythroid cell lineage, consistent with the clinical phenotype of RPS14 or RPS19 haploinsufficiency. Moreover, immunohistochemical analysis of bone marrow biopsies from patient with the 5q- syndrome demonstrated intense staining of TP53 that was restricted to erythroid progenitor cells. Taken together our study indicates that inhibition of ribosomal biogenesis causes TP53 activation selectively in erythroid progenitor cells. Clinically, TP53 staining of patient samples could be used as a diagnostic marker for some types of MDS. Disclosures: No relevant conflicts of interest to declare.

PKA Mediated Phosphorylation of TAL1 Regulates Its Interaction with LSD1 During Hematopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2599-2599
Author(s):  
Ying Li ◽  
Changwang Deng ◽  
Xin Hu ◽  
Xueqi Fu ◽  
Yi Qiu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Transcriptional Activation ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Functional Link ◽  
Dynamic Regulation ◽  
Helix Loop Helix ◽  
Interacting Domain

Abstract Abstract 2599 TAL1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages. TAL1 is a phosphorylated protein and its activities are mediated by the corepressors and coactivators that associate with TAL1. However, the functional link between phosphorylation and the recruitment of co-regulators by TAL1 is currently unknown. We showed that TAL1 dynamically interacts with LSD1 complex containing both histone H3K4 demethylase and deacetylase activities during hematopoiesis (Proc. Natl. Acad. Sci. USA 106: 10141–10146). To further understand the molecular mechanism that regulates the TAL1 and LSD1 interaction during hematopoiesis, we determined whether TAL1 directly interacts with LSD1 and characterized the domains required for this interaction. TAL1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142–185 proximal to the bHLH domain, which contains a serine 172 residue that becomes phosphorylated by Protein kinase A (PKA) during the transcriptional activation of TAL1. The PKA inhibitor, H89, stimulated TAL1 interaction with LSD1 in hematopoietic cells, while Forskolin, an activator of PKA, completely abolished TAL-LSD1 interaction. We further mutated serine 172 of TAL1 to Alanine (Ala) or to Aspartic acid (Asp) to mimic unphosphorylated or phosphorylated TAL1, respectively. While the TAL1Ser172Ala mutant remains interacted with LSD1, TAL1Ser172Asp specifically loses its interaction with LSD1 indicating that serine 172 phosphorylation of TAL1 by PKA destabilizes the TAL1 and LSD1 interaction. Our previous results indicate that LSD1 inhibits TAL1 mediated erythroid differentiation. To further test whether the activity of TAL1 is mediated through an interaction with LSD1, we expressed the TAL1 mutant that deleted the LSD1 interacting domain in erythroid progenitor cells and showed that the deletion of the LSD1 interacting domain of TAL1 leads to a promotion of erythroid differentiation and H3K4 hypermethylation of the P4.2 promoter. In contrast, the expression of the TAL1Ser172Ala mutant and TAL1-LSD1 chimerical fusion enhanced cellular proliferation and colony formation ability of the hematopoietic progenitor cells while these constructs inhibited erythroid differentiation. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and erythroid differentiation. The results suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs. Disclosures: No relevant conflicts of interest to declare.

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2674-2674
Author(s):  
Naoya Uchida ◽  
Atsushi Fujita ◽  
Thomas Winkler ◽  
John F. Tisdale
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Ips Cells ◽  
Cell Fraction ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Ips Cell ◽  
Erythroid Progenitor Cells ◽  
Spherical Cells ◽  
Induced Pluripotent

Abstract Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell (RBC) transfusion. When ES cell-derived erythroid cells are generated using embryoid bodies, these cells predominantly express embryonic type ε-globin, with lesser fetal type γ-globin and small amounts of adult type β-globin; however, no β-globin expression is detected in iPS cell-derived erythroid cells. Recently, the ES cell-derived sac (ES sac) was reported to express hemangioblast markers and could generate functional platelets (Takayama, Blood. 2008). We previously demonstrated that erythroid cells were also efficiently generated via the ES sac (2013 ASH). We extend this work to evaluate globin expression in ES sac-derived erythroid cells. We generated ES sacs from human H1 ES or iPS cells using VEGF for 15 days, as previously described. The spherical cells within ES sacs were harvested and cultured on OP9 feeder cells for 2 days, and the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002). The globin types expressed in erythroid cells were evaluated by RT-qPCR and hemoglobin electrophoresis. When hematopoietic cell-stimulating cytokines (SCF, FLT3L, TPO, IL3, EPO, and BMP4) were added in ES sac cultures on day 9-15, we observed 1.4-fold greater amounts of GPA+ erythroid cells (p<0.05) and 1.3-fold lower ε-globin expression in ES sac-derived erythroid cells (p<0.05), suggesting that cytokine stimulation might induce more hematopoietic/stem progenitor cells (HSPC) which can be differentiated to γ- or β-globin-expressing erythroid cells. Thus, we hypothesized that the ES sac contains both primitive and definitive erythroid progenitor cells capable of ε-globin-expression or γ- or β-globin-expression upon differentiation; respectively, and that these progenitors are selectable based upon surface markers of erythroid progenitor cells or HSPCs. To investigate whether primitive erythropoiesis is switched to definitive erythropoiesis during ES sac maturation, we evaluated spherical cells within the ES sac on day 9, 12, 15, and 18 after ES sac culture. A high percentage of GPA+ erythroid cells (29.2±3.7%) were observed on as early as day9. At that time point, almost no CD34+CD45+ HSPCs were present; however, the number increased upon further ES sac maturation until day 15 (6.8±1.6%). Cells further differentiated in erythroid culture had lower ε-globin expression and higher β-globin expression (up to 13.8±1.5%) when harvested from the ES sac at later time points. These data suggest that more matured ES sacs favor less primitive erythropoiesis and more definitive erythropoiesis. On day 15, the ES sacs contained a high percentage of GPA+(CD34-) erythroid cells (68.7±4.0%) and relatively lower amounts of CD34+(GPA-) HSPCs (16.7±2.1%). Therefore, we separated GPA+ and GPA- spherical cells from ES sac by magnetic selection before further erythroid differentiation, which resulted in higher ε-globin expression (43.0±16.6% vs 4.4±1.2%, p<0.01) and lower β-globin expression (7.6±5.3x10e-7% vs 19.8±2.7%, p<0.01) from the GPA+ cell fraction. In contrast, after erythroid differentiation from CD34+ or CD34- sorted spherical cells, lower ε-globin expression (3.7±0.3% vs 17.1±0.9%, p<0.01) and higher β-globin expression (17.4±0.7 % vs 0.9±0.4 %, p<0.01) were observed from the CD34+ cell fraction. These data suggest that the ES sac contains both primitive erythroid progenitor cells in the CD34- or GPA+ cell fraction and definitive erythroid progenitor cells in the CD34+ or GPA- cell fraction. In addition, iPS sac-derived erythroid cells were generated from 2 clones of fibroblast-derived iPS cells, which demonstrated 9.0±2.6% (clone #1) and 7.3±3.7% (clone #2) of β-globin expression. These data demonstrate that similar to ES sac-derived erythroid cells, iPS cell-derived erythroid cells can produce β-globin when differentiated from iPS sacs. In conclusion, we demonstrate that human ES and iPS cells can generate both primitive and definitive erythroid progenitor cells when differentiated in ES/iPS sac. CD34 or GPA discriminates between primitive and definitive erythroid progenitor cells in ES sac. The presented differentiation and selection strategy represent an important step to develop in vitro RBC production system from pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Potent but transient immunosuppression of T-cells is a general feature of erythroid progenitor cells

2021 ◽  
Author(s):  
Tomasz M. Grzywa ◽  
Anna Sosnowska ◽  
Zuzanna Rydzynska ◽  
Michal Lazniewski ◽  
Dariusz Plewczynski ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Early Stage ◽  
Erythroid Differentiation ◽  
Peripheral Blood Mononuclear ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

AbstractErythroid progenitor cells (EPCs) have been recently recognized as potent immunoregulatory cells with defined roles in fetomaternal tolerance and immune response to infectious agents in neonates and cancer patients. Here, we show that early-stage EPCs are enriched in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). EPCs expansion in anemic mice leads to the L-arginine depletion in the spleen microenvironment resulting in the suppression of T-cell responses. In humans with anemia, EPCs expand and express both ARG1 and ARG2 that participate in suppressing the proliferation and production of IFN-γ from T-cells. EPCs differentiated from peripheral blood mononuclear cells potently suppress T-cell proliferation and this effect is the most prominent for CD49dhi CD71hiEPCs. The suppressive properties disappear during erythroid differentiation as more differentiated EPCs as well as mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of EPCs in the regulation of immune response.Abstract Figure

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