Leukemia Cell Infiltration Causes Defective Erythropoiesis Partially through MIP-1alpha

Abstract Leukemia often results in severe anemia, which may significantly contributes to mortality and morbidity of the patients. However, the mechanisms underlying the insufficient erythropoiesis in leukemia have been poorly understood. In this study, with our recently established non-irradiated MLL-AF9 acute myeloid leukemia (AML) murine model (Cheng H et al, Blood 2015), we observed a significant decrease in hemoglobin and red blood cells (RBCs) of Peripheral blood (PB) in the leukemic mice (n=6 per group, p=0.0122 vs p=0.0003). The absolute numbers of the erythroblasts at different stages (Pro Es, Ery.A, Ery.B, Ery.C) in bone marrow (BM) were also reduced. Consistently, by gene set enrichment analysis (GSEA) of microarray data of LKS+ cells (GSE52506 ) from leukemic mice, we found significant down-regulation of erythroid differentiation related genes such as GATA1, FOG-1, LMO2 and KLF1. These genes were more significantly inhibited in megakaryocytic-erythroid progenitors (MEPs) and Pro Es from the leukemic mice. Notably, the MEPs were the most reduced subset among all the committed hematopoietic progenitor cells (HPCs) during leukemia progression (90% decrease compared to control, p = 0.0007). MEPs were gradually accumulated in the G0 phase (from 22% to 70%, p<0.001). In contrast, erythroblasts (Pro Es, Ery.A, Ery.B) were more cycling (G1/S/G2/M) in leukemic mice and the proportions of Annexin V+ cells in erythroblasts but not in MEPs were also increased during leukemia development. Colony-forming cell (CFC) assays revealed that BM plasma of leukemia mice exerted an inhibitory effect on both BFU-Es and CFU-Es of BM mononuclear cells (BMMNCs) but not on other types of colonies (40% decrease for CFU-Es, 60% decrease for BFU-Es, p<0.001). Consistently, BM plasma of AML patients could also reduce the yield of BFU-Es and CFU-Es from CD34+ cord blood cells (n=7, p=0.006). To determine which cytokines may be responsible for the inhibitory effect, we collected serum and BM plasma from control and leukemic mice for cytokine array analysis. Among the elevated cytokines, MIP-1alpha was previously reported to be up-regulated in leukemic stem cells and its higher expression was found in the majority of patients with leukemia and a subset of patients with lymphoma and myeloma according to the Oncomine data set. We also confirmed it in a cohort of untreated AML patients (n=32). Importantly, AML patients with higher expression of MIP-1alpha showed reduced survival time (median=13.08 months) compared with the patients with lower expression (median=25.86 months) based on the leukemia-gene-atlas (LGA) analysis (n=72). By the CFC assay and single cell culture with different subsets of hematopoietic stem cells (HSCs) and HPCs, MIP-1alpha was able to largely mimic the inhibitory effect on the erythroid differentiation at both stem cell and progenitor cell levels. Mechanistically, we observed higher expression of MIP-1alpha receptor CCR1 in HSCs, MEPs and erythroblasts than CCR5. Administration of CCR1 antagonist, BX471 could partially recover the yield of erythroid colonies after treatment of MIP-1alpha or leukemia BM plasma. An increase of phosphorylation of p38 (phos p38) and resulted down-regulation of GATA1 after MIP-1alpha treatment were documented by Western blots and immunostaining. In summary, our results demonstrate that leukemic cell infiltration causes severe inhibition of erythropoiesis largely at different erythroid precursor levels and this inhibitory effect is at least partially medicated by elevated MIP-1alpha level via CCR1-p38 activation in the leukemic microenvironment. Disclosures No relevant conflicts of interest to declare.

Download Full-text

In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

BioMed Research International ◽

10.1155/2013/807863 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 7

Author(s):

Jiafei Xi ◽

Yanhua Li ◽

Ruoyong Wang ◽

Yunfang Wang ◽

Xue Nan ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Red Blood Cells ◽

Stromal Cells ◽

Blood Cells ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

Download Full-text

ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with the Stat5b Pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e10-01-0040 ◽

2010 ◽

Vol 21 (19) ◽

pp. 3340-3351 ◽

Cited By ~ 19

Author(s):

Tatiana Vignudelli ◽

Tommaso Selmi ◽

Andrea Martello ◽

Sandra Parenti ◽

Alexis Grande ◽

...

Keyword(s):

Stem Cells ◽

Untranslated Region ◽

Target Genes ◽

Cumulative Effect ◽

Erythroid Differentiation ◽

Myeloproliferative Disorders ◽

Hematopoietic Differentiation ◽

Hematopoietic Progenitors ◽

Down Regulation ◽

Hematopoietic Stem

ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3′-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by posttranscriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3′ untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, because cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders.

Download Full-text

Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol

Blood ◽

10.1182/blood-2003-07-2414 ◽

2004 ◽

Vol 103 (10) ◽

pp. 3710-3716 ◽

Cited By ~ 61

Author(s):

Peter A. Horn ◽

Kirsten A. Keyser ◽

Laura J. Peterson ◽

Tobias Neff ◽

Bobbie M. Thomasson ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Gene Transfer ◽

Peripheral Blood ◽

Blood Cells ◽

Lentiviral Vectors ◽

Large Animal ◽

Hematopoietic Stem ◽

Lentiviral Transduction ◽

Mobilized Peripheral Blood

Abstract The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34+ hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)– and granulocyte-colony stimulating factor (G-CSF)–primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.

Download Full-text

Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽

10.1182/blood-2020-137426 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-31

Author(s):

Maria Rosa Lidonnici ◽

Giulia Chianella ◽

Francesca Tiboni ◽

Matteo Barcella ◽

Ivan Merelli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Gene Set Enrichment Analysis ◽

Lineage Commitment ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Hematopoietic Stem ◽

Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Merocyanine 540-sensitized photoinactivation of human erythrocytes parasitized by Plasmodium falciparum

Blood ◽

10.1182/blood.v80.1.21.bloodjournal80121 ◽

1992 ◽

Vol 80 (1) ◽

pp. 21-24 ◽

Cited By ~ 1

Author(s):

OM Smith ◽

SA Dolan ◽

JA Dvorak ◽

TE Wellems ◽

F Sieber

Keyword(s):

Stem Cells ◽

Plasmodium Falciparum ◽

Hematopoietic Stem Cells ◽

Red Blood Cells ◽

Human Blood ◽

Blood Cells ◽

Potential Usefulness ◽

Hematopoietic Stem ◽

Infected Blood ◽

Merocyanine 540

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine- sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.

Download Full-text

MicroRNAs as Haematopoiesis Regulators

Advances in Hematology ◽

10.1155/2013/695754 ◽

2013 ◽

Vol 2013 ◽

pp. 1-20 ◽

Cited By ~ 46

Author(s):

Ram Babu Undi ◽

Ravinder Kandi ◽

Ravi Kumar Gutti

Keyword(s):

Blood Cells ◽

Erythroid Differentiation ◽

Lymphocytic Leukemia ◽

Myelogenous Leukemia ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Complex Process ◽

Development And Differentiation ◽

Multiple Myelomas

The production of different types of blood cells including their formation, development, and differentiation is collectively known as haematopoiesis. Blood cells are divided into three lineages erythriod (erythrocytes), lymphoid (B and T cells), and myeloid (granulocytes, megakaryocytes, and macrophages). Haematopoiesis is a complex process regulated by several mechanisms including microRNAs (miRNAs). miRNAs are small RNAs which regulate the expression of a number of genes involved in commitment and differentiation of hematopoietic stem cells. Evidence shows that miRNAs play an important role in haematopoiesis; for example, myeloid and erythroid differentiation is blocked by the overexpression of miR-15a. miR-221, miR-222, and miR-24 inhibit the erythropoiesis, whereas miR-150 plays a role in B and T cell differentiation. miR-146 and miR-10a are downregulated in megakaryopoiesis. Aberrant expression of miRNAs was observed in hematological malignancies including chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myelomas, and B cell lymphomas. In this review we have focused on discussing the role of miRNA in haematopoiesis.

Download Full-text

Megakaryocytic differentiation of CD133+ hematopoietic stem cells by down-regulation of microRNA-10a

Hematology ◽

10.1179/1607845412y.0000000047 ◽

2013 ◽

Vol 18 (2) ◽

pp. 93-100 ◽

Cited By ~ 10

Author(s):

M. Nikougoftar Zarif ◽

M. Soleimani ◽

H. Abolghasemi ◽

N. Amirizade ◽

E. Arefian ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Down Regulation ◽

Hematopoietic Stem ◽

Megakaryocytic Differentiation

Download Full-text

Down-regulation of GATA1 uncouples STAT5-induced erythroid differentiation from stem/progenitor cell proliferation

Blood ◽

10.1182/blood-2009-10-250894 ◽

2010 ◽

Vol 115 (22) ◽

pp. 4367-4376 ◽

Cited By ~ 16

Author(s):

Albertus T. J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Target Genes ◽

Lentiviral Vector ◽

Erythroid Differentiation ◽

Down Regulation ◽

Hematopoietic Stem ◽

Progenitor Cell Proliferation ◽

Cord Blood Cells

Abstract Previously, we have shown that overexpression of an activated mutant of signal transducer and activator of transcription-5 (STAT5) induces erythropoiesis, impaired myelopoiesis, and an increase in long-term proliferation of human hematopoietic stem/progenitor cells. Because GATA1 is a key transcription factor involved in erythropoiesis, the involvement of GATA1 in STAT5-induced phenotypes was studied by shRNA-mediated knockdown of GATA1. CD34+ cord blood cells were double transduced with a conditionally active STAT5 mutant and a lentiviral vector expressing a short hairpin against GATA1. Erythropoiesis was completely abolished in the absence of GATA1, indicating that STAT5-induced erythropoiesis is GATA1-dependent. Furthermore, the impaired myelopoiesis in STAT5-transduced cells was restored by GATA1 knockdown. Interestingly, early cobblestone formation was only modestly affected, and long-term growth of STAT5-positive cells was increased in the absence of GATA1, whereby high progenitor numbers were maintained. Thus, GATA1 down-regulation allowed the dissection of STAT5-induced differentiation phenotypes from the effects on long-term expansion of stem/progenitor cells. Gene expression profiling allowed the identification of GATA1-dependent and GATA1-independent STAT5 target genes, and these studies revealed that several proliferation-related genes were up-regulated by STAT5 independent of GATA1, whereas several erythroid differentiation-related genes were found to be GATA1 as well as STAT5 dependent.

Download Full-text

Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells

Blood ◽

10.1182/blood.v83.12.3758.3758 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3758-3779 ◽

Cited By ~ 123

Author(s):

N Uchida ◽

HL Aguila ◽

WH Fleming ◽

L Jerabek ◽

IL Weissman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Critical Role ◽

White Blood Cells ◽

Cell Types ◽

Dose Dependence ◽

Hematopoietic Stem ◽

Hematopoietic Recovery

Abstract Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca- 1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

Download Full-text

MLL leukemia induction by t(9;11) chromosomal translocation in human hematopoietic stem cells using genome editing

Blood Advances ◽

10.1182/bloodadvances.2017013748 ◽

2018 ◽

Vol 2 (8) ◽

pp. 832-845 ◽

Cited By ~ 16

Author(s):

Corina Schneidawind ◽

Johan Jeong ◽

Dominik Schneidawind ◽

In-Suk Kim ◽

Jesús Duque-Afonso ◽

...

Keyword(s):

Stem Cells ◽

Genome Editing ◽

Blood Cells ◽

Chromosomal Translocation ◽

Chromosomal Translocations ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Key Points ◽

Cord Blood Cells

Key Points Genome editing induces t(9;11) chromosomal translocations and transforms primary CD34+ human cord blood cells leading to acute leukemia. CD9 is upregulated in primary t(9;11) cells and is a useful marker for enrichment of genome-edited MLL-rearranged cells in vitro.

Download Full-text