c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression

Blood ◽  
2010 ◽  
Vol 116 (22) ◽  
pp. e99-e110 ◽  
Author(s):  
Elisa Bianchi ◽  
Roberta Zini ◽  
Simona Salati ◽  
Elena Tenedini ◽  
Ruggiero Norfo ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Luciferase Reporter ◽  
Myb Transcription Factor ◽  
Promoter Regions ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb–silenced CD34+ cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

scholarly journals Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2812-2821 ◽  
Author(s):  
Fabiana Perna ◽  
Nadia Gurvich ◽  
Ruben Hoya-Arias ◽  
Omar Abdel-Wahab ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Myeloproliferative Neoplasms ◽  
Erythroid Differentiation ◽  
Polycomb Group ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract L3MBTL1, the human homolog of the Drosophila L(3)MBT polycomb group tumor suppressor gene, is located on chromosome 20q12, within the common deleted region identified in patients with 20q deletion-associated polycythemia vera, myelodysplastic syndrome, and acute myeloid leukemia. L3MBTL1 is expressed within hematopoietic CD34+ cells; thus, it may contribute to the pathogenesis of these disorders. To define its role in hematopoiesis, we knocked down L3MBTL1 expression in primary hematopoietic stem/progenitor (ie, CD34+) cells isolated from human cord blood (using short hairpin RNAs) and observed an enhanced commitment to and acceleration of erythroid differentiation. Consistent with this effect, overexpression of L3MBTL1 in primary hematopoietic CD34+ cells as well as in 20q− cell lines restricted erythroid differentiation. Furthermore, L3MBTL1 levels decrease during hemin-induced erythroid differentiation or erythropoietin exposure, suggesting a specific role for L3MBTL1 down-regulation in enforcing cell fate decisions toward the erythroid lineage. Indeed, L3MBTL1 knockdown enhanced the sensitivity of hematopoietic stem/progenitor cells to erythropoietin (Epo), with increased Epo-induced phosphorylation of STAT5, AKT, and MAPK as well as detectable phosphorylation in the absence of Epo. Our data suggest that haploinsufficiency of L3MBTL1 contributes to some (20q−) myeloproliferative neoplasms, especially polycythemia vera, by promoting erythroid differentiation.

scholarly journals Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Gordon G. L. Wong ◽  
Gabriela Krivdova ◽  
Olga I. Gan ◽  
Jessica L. McLeod ◽  
John E. Dick ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Erythroid Precursor ◽  
Lineage Differentiation ◽  
Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

scholarly journals Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1218-1227 ◽  
Author(s):  
LW Terstappen ◽  
S Huang ◽  
M Safford ◽  
PM Lansdorp ◽  
MR Loken
Keyword(s):  
Progenitor Cells ◽  
Lineage Commitment ◽  
Multiparameter Flow Cytometry ◽  
Differentiation Markers ◽  
Single Class ◽  
Hematopoietic Stem ◽  
Cd34 Antigen ◽  
Cd34 Cells ◽  
Cell Fractions ◽  
Cd38 Antigen

Abstract Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.

MicroRNA hsa-mir-16 Contributes to Regulation of Myeloid Differentiation of Human CD34+ Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1343-1343
Author(s):  
Richard Hildreth ◽  
Robert W. Georgantas ◽  
Roshan Patel ◽  
Sebastien Morisot ◽  
Jonathan Alder ◽  
...  
Keyword(s):  
Protein Expression ◽  
K562 Cells ◽  
Negative Regulator ◽  
Luciferase Reporter ◽  
Myeloid Differentiation ◽  
Expression Data ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract In a large microRNA-array and bioinformatics study, we determined all of the microRNAs (miRs) expressed by human CD34+ hematopoietic stem-progenitor cells (HSPCs) from bone marrow and G-CSF mobilized blood. When we combined miR expression data, mRNA expression data fro a previous study (Georgantas et al, Cancer Research 64:4434), and data from various published mir-target prediction algorithms, we were able to bioinformaticly predict the actions of miRs within the hematopoietic system. MicroRNA hsa-mir-16 was highly expressed in CD34+ HSPCs, and was predicted to target several HSPC-expressed mRNAs (CXCR4, HoxB7, Runx-1, ETS-1, and Myb) that encode proteins known to be critically involved specifically in myelopoiesis within the hematopoietic system. We first confirmed that protein expression from each of these putative target mRNAs was in fact regulated by mir-16. The 3′UTR sequence from each of these mRNAs was cloned behind a luciferase reporter. Each reporter construct was transfected into K562 cells, which strongly express mir-16. In all cases, protein expression from the predicted target mRNA was greatly reduced in K562 cells, as compared to controls. As a first determination of mir-16’s function in hematopoietic cells, HL60 and K562 cells were transduced with hsa-mir-16 lentivirus, then treated with various chemical differentiation inducers. As was predicted by bioinformatics, hsa-mir-16 halted myeloid differentiation of HL60 cells, but did not affect megakaryocytic differentiation or erythroid differentiation of K562 cells. These initial findings suggest that mir-16 is a specific negative regulator of myelopoiesis. We are currently evaluating the effects of mir-16 on normal human CD34+ cells by in vitro CFC and suspension culture assays, as well as in vivo by transplantation of hsa-mir-16 lentivirus transduced cells in immunodeficient mice.

MicroRNA-486-5p Targets Foxo1 and Regulates Human Hematopoietic Stem Cell Proliferation and Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3871-3871
Author(s):  
Li-Sheng Wang ◽  
Ling LI ◽  
Liang Li ◽  
Keh-Dong Shiang ◽  
Min Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Myeloid Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Control Vector ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Direct Target

Abstract Abstract 3871 Previous studies have supported a critical role for specific miRNA in regulating hematopoiesis. However the relative abundance and specificity for most miRNAs remains to be investigated, and the role of expressed miRNA in regulating cell fate and function remains poorly understood. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. miR-486 is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is reported to be enriched in muscle cells. The expression pattern of miR-486 in hematopoietic cells and its roles in hematopoietic regulation are not known. In CB cells, miR-486 expression level was highest in MEP and was low in HSC. There was 16-fold increased expression of miR-486 during in vitro erythroid differentiation of CB Lin-CD34+CD38– cells, associated with 5-fold increase in Ankyrin-1 gene expression. To explore the role of miR-486 in growth and differentiation of hematopoietic stem and progenitor cells (HSPC), we first expressed hsa-miR-486-5p in CB CD34+ cells using lentiviral vectors. CB CD34+ cells transduced with this vector demonstrated 2–3 fold increased expression of miRNA-486-5p compared to cells transduced with a control vector (Ctrl). CB CD34+ cells expressing miR-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days. Increased numbers of erythroid cells and reduced numbers of myeloid cells were generated in culture (GPA+ cells: Ctrl 58% and miR-486-5p 72.2%; CD33+ cells: Ctrl 30.7% and miR-486-5p 21.9%;, CD11b cells: Ctrl 33.5% and miR-486-5p 21.5%). To further investigate the effect of inhibition of miR-486-5p on growth and differentiation of HSPC, we inhibited miR-486 expression in CB CD34+ cells using a modified miRZip anti-miRNA lentivirus vectors (SBI) expressing anti-miR-486-5p and compared to cells expressing a control scrambled anti-miRNA sequence. Anti-miR-486-5p expressing CB CD34+ cells generated reduced number of cells in growth factor (GF) culture (67.5% inhibition) compared to controls. Greater inhibition of erythroid compared to myeloid cells was seen (GPA+ cells: 62.5% inhibition; CD33+ cells: 37.1% inhibition compared to controls at day 6). Anti-miR-486-5p expressing CB CD34+ cells also demonstrated reduced colony formation (BFU-E: 67% inhibition;, CFU-GM 16% inhibition), and reduced proliferation (43.88% inhibition of proliferation index) compared to controls. Similar results were observed with CB Lin-CD34+CD38- cells transduced with anti-486-5p virus (GPA+ cells: 67% inhibition; CD33+ cells: 30 % inhibition). The number of CD34+ cells was however maintained after culture (117% for miR-486-5p compared to scramble). These results indicate an important role for miR-486-5p in preservation, proliferation and erythroid differentiation of HSC. A search for evolutionarily conserved miR-486-5p targets using Targetscan 5.1 identified Foxo1, a member of the Foxo subfamily of forkhead transcription factors which play negative regulatory roles in hematopoiesis, as the highest ranking target. To demonstrate that Foxo1 is a direct target of miR-486-5p, we generated pMIR-REPORT™ constructs containing two miR-486-5p seed sites (182 and 658) within the Foxo1 3′-UTR. These constructs were cotransfected into HEK293T cells along with a miR-486-5p expression plasmid or empty control vector. Expression of miR-486-5p resulted in a 65% reduction in luciferase activity. Expression of anti-miR-486-5p resulted in increased Foxo1 protein expression in CB CD34+ cells. Expression of miR-486-5p also resulted in 50% decrease of Foxo1 protein expression. Using a Fas-L promoter-luciferase reporter we found that inhibition of miR486-5p increased Foxo1 transactivation activity in HEK293T cells. These results demonstrate that Foxo1 is a direct target of miR-486-5p. We conclude that miR-486-5p expression is modulated during normal hematopoietic differentiation and in leukemic hematopoiesis. Our results indicate a regulatory role for miR-486-5p in the growth hematopoietic stem cells and their erythroid differentiation. We show that miR-486-5p directly inhibits Foxo1 expression, which may potentially play an important role in its hematopoietic regulatory function. Disclosures: No relevant conflicts of interest to declare.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4107-4107
Author(s):  
Susan Hilgendorf ◽  
Hendrik Folkerts ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Erythroid Differentiation ◽  
Lentiviral Vectors ◽  
Apoptotic Response ◽  
Double Positive ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p<0.01) and, to some extent, granulocytic and macrophage colony formation as well (p<0.09, p<0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p<0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1218-1227 ◽  
Author(s):  
LW Terstappen ◽  
S Huang ◽  
M Safford ◽  
PM Lansdorp ◽  
MR Loken
Keyword(s):  
Progenitor Cells ◽  
Lineage Commitment ◽  
Multiparameter Flow Cytometry ◽  
Differentiation Markers ◽  
Single Class ◽  
Hematopoietic Stem ◽  
Cd34 Antigen ◽  
Cd34 Cells ◽  
Cell Fractions ◽  
Cd38 Antigen

Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

scholarly journals Adult Donor-Derived Human CD34+ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4378-4378
Author(s):  
Yasuyuki Saito ◽  
Jana M. Ellegast ◽  
Rouven Müller ◽  
Richard A. Flavell ◽  
Markus G. Manz
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Organs ◽  
Humanized Mice ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Lymphoid System ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Transplantation of human CD34+ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, adult donor-derived CD34+ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3rd generation humanized mice (3rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2-/-γC-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014). To evaluate human adult CD34+ cell engraftment in 3rd gen. huMice, CD34+ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x105 cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γC-/- (NSG) mice or Rag2-/-γC-/-hSirpαTg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45+ cell engraftment (>1% of total CD45+ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46+ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34+ and CD34+CD38- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45+ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice. Taken together, this study demonstrates that our 3rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals. Disclosures No relevant conflicts of interest to declare.

scholarly journals In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4040-4048 ◽  
Author(s):  
M Rosenzweig ◽  
DF Marks ◽  
H Zhu ◽  
D Hempel ◽  
KG Mansfield ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
T Cell Differentiation ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

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