CD133 Expression on ALL Progenitor Cells: Further Evidence for a Hierarchy within the Leukaemic Clone.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 523-523
Author(s):  
Charlotte V. Cox ◽  
Roger S. Evely ◽  
Nicholas J. Goulden ◽  
Allison Blair
Keyword(s):  
Progenitor Cells ◽  
Significant Proportion ◽  
Lymphoid Cells ◽  
Stem Cell Marker ◽  
Proliferating Cells ◽  
Cell Numbers ◽  
Proliferative Ability

Abstract Despite important advances in the refinement of therapy for acute lymphoblastic leukaemia (ALL), a significant proportion, ~30%, relapse due to failure to eradicate the disease. ALL is thought to be maintained by a subpopulation of cells with extensive proliferative capacity, unlike the majority of blasts, which have limited proliferative ability. This sub-population may be resistant to drug regimens designed to kill the bulk ALL population and subsequent relapses may arise from these cells. Hence, identification and characterisation of these putative ALL stem cells is essential for the development of more effective therapeutic strategies. We have demonstrated that ALL cells capable of long-term proliferation in vitro and in vivo are CD34+/CD10−/CD19−, suggesting that cells with an immature phenotype, rather than committed B-lymphoid cells, are the targets for transformation in B cell leukaemias. Here we have attempted to further define these ALL progenitor cells by investigating the expression of CD133, the stem cell marker, on cells with long-term proliferative ability in vitro and in vivo. ALL cells from 6 pts at diagnosis (4 c-ALL, 2 pre B) and 3 c-ALL in relapse were sorted for expression of CD133 and CD19 and evaluated in a suspension culture (SC) assay. The majority of cells at sorting were CD133−/CD19+ (59±7%) and the CD133+/CD19+ and CD133+/CD19− subfractions represented only (9±6%) and (0.8±0.3%) respectively. However, after 3 weeks in SC, the majority of cells were derived from the CD133+/CD19− subfraction (62±8%). This trend continued with 77±7% of proliferating cells derived from the CD133+/CD19− subfraction by week 6. In the cultures of CD133+/CD19− cells there was a 4 - > 4 log fold expansion in cell numbers, starting from an average 9.3x104 cells at initiation to an average 1.8x106 cells at week 6. Unsorted cells and cells sorted for expression of CD133 and CD19 from these 9 pts were evaluated for their ability to repopulate sublethally irradiated NOD/SCID mice. Engraftment was achieved in each case using unsorted cells (0.7–38% CD45+, with 2x106–107 cells). The only sorted subfraction that engrafted were CD133+/CD19− cells (range 0.8–70% CD45+, using 103–5x104 cells). There was no engraftment with the other subfractions despite injecting significantly higher cell numbers. Secondary transplantation experiments to evaluate the self-renewal potential of the CD133+/CD19− cells are ongoing. Cytogenetic analyses of CD133+/CD19− cells have revealed that they contain translocations such as TEL-AML1 suggesting the translocations occurred as early leukaemogenic events rather than as the blast cells differentiate. IgH rearrangements present in the bulk ALL population at diagnosis were also detected in the CD133+/CD19− cells by PCR analyses. These data suggest that ALL cells with long-term proliferative ability and NOD/SCID repopulating ability express CD133 and lack expression of CD19 providing further evidence for the existence of a hierarchy of progenitor cells in ALL. More precise definition of these ALL progenitor cells should improve MRD immunosurveillance techniques, that are based on the phenotype of the total cell population and may not detect the leukaemic progenitor cells, and permit investigation of the efficacy of therapeutic agents on the cells that may be responsible for disease relapse.

Stem Cells in T-ALL Have a Primitive CD133+/CD34+/CD7- Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1885-1885
Author(s):  
Charlotte V. Cox ◽  
Roger S. Evely ◽  
Nicholas J. Goulden ◽  
Allison Blair
Keyword(s):  
Stem Cells ◽  
Cell Types ◽  
Scid Mice ◽  
Cell Of Origin ◽  
Proliferating Cells ◽  
Self Renewal ◽  
Proliferative Ability

Abstract The cell of origin of childhood acute lymphoblastic leukaemia (ALL) has been the subject of conflicting reports in recent years. One model suggests that many haemopoietic cell types are susceptible to transformation and the level of commitment of the target cell influences the characteristics of the resulting blast cell population. A second model suggests that primitive haemopoietic cells are the targets for transformation, with some differentiation occurring subsequent to the transformation event. This model suggests a hierarchy of progenitors may exist in ALL. In support of this latter model, we have demonstrated that leukaemic stem cells in B-ALL have a primitive CD34+/CD10−/CD19− phenotype and T-ALL cells with NOD/SCID engrafting capacity are CD34+/CD4−. In this investigation we have attempted to further purify and characterise leukaemic stem cells from children with T-ALL. Cells from 7 patients were sorted for expression of CD34 and CD7 and the sorted subfractions evaluated for long-term proliferative ability in vitro using a serum free suspension culture assay and in the NOD/SCID mouse model. In this group of patients, the CD34+/CD7+ fraction represented 7±6% of cells at sorting, 6±4% were CD34+/CD7− and the majority were CD34−/CD7+ (60±12%). After 3 weeks in culture, the majority of proliferating cells were derived from the CD34+/CD7− subfraction (53±16%). By week 6, >70% of proliferating cells were derived from the CD34+/CD7− subfraction. Unsorted ALL cells and the sorted subfractions from 4 of these patients, were evaluated for their ability to engraft sublethally irradiated NOD/SCID mice. In each case, engraftment was achieved using 105–106 unsorted cells (25–80% CD45+) and with the CD34+/CD7− subfraction only (4–84% CD45+ with 3x103–8x104 cells). There was no engraftment with the other subfractions despite injecting up to 100 fold more cells. The engrafted cells had the same karyotype as the patient at diagnosis and expressed high levels of CD2, CD4 and CD7 implying they had differentiated in vivo. The self-renewal capacity of the CD34+/CD7− cells was evaluated by secondary transplantation. CD45+ cells from NOD/SCIDs engrafted with CD34+/CD7− cells successfully engrafted secondary recipients with equivalent levels of human cell engraftment, demonstrating these cells were capable of self-renewal. These findings suggest that cells with a more primitive phenotype may be the targets for transformation in T-ALL, rather than committed lymphocytes. To further investigate this hypothesis, we sorted cells from 4 of these patients for expression of CD133 and CD7 and evaluated their proliferative ability as described above. Results to date indicate that the CD133+/CD7− fraction represents only 0.35% of nucleated cells at sorting. However, after 3 weeks in culture, 48±9% of proliferating cells were derived from this subfraction and by week 6, 58±20% of cells were derived from the CD133+/CD7− subfraction. In vivo analyses completed in 2 patients to date have shown that only the CD133+/CD7− subfraction was capable of engrafting NOD/SCID mice (0.5–54% CD45+ using 3x103–105 cells). These results demonstrate that T-ALL cells with long-term proliferative and NOD/SCID repopulating capacity express the primitive haemopoietic cell antigens CD133 and CD34 and lack expression of T-lineage markers. These findings add further support to the concept of a common cell of origin for acute leukaemias.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

Effect on the Sensitivity of Lymphoid Cells to Acriflavine after « in vivo » Treatment with Heterologous Albumin Coupled with Diazotized Acriflavin

1966 ◽  
Vol 52 (3) ◽  
pp. 177-185
Author(s):  
Aurelio Di Marco ◽  
Rosella Silvestrini ◽  
Emidio Calendi
Keyword(s):  
Lymph Nodes ◽  
Proliferative Activity ◽  
Lymphoid Cells ◽  
Low Doses ◽  
Proliferating Cells ◽  
In Vivo Treatment ◽  
Albino Mice ◽  
Different Doses

The possibility that the «in vivo» treatment with heterologous albumin coupled with diazotized acriflavine may affect the sensitivity of lymphoid cells to the action of acriflavine was studied. Albino mice CFW strain were treated subcutanceusly with the coupled albumin in the presence of complete Freund adjuvant. Lymph nodes from control and immunized animals, fifteen days after the treament, were cultured «in vitro» in the presence of different doses of acriflavine (from 0.5 to 4 μg/ml). The action of acriflavine was evaluated as the growth of cultures, the percent of lymphoid cells in the different phases of differentiation and the percent of proliferating cells after incubation for 24 hours in the presence of 3H thymidine. Results show that lymphoid cells of immunized mice are less sensitive to the citotoxic activity of acriflavine than those of the controls. Acriflavine, at low doses, reduces the growth of normal cultures and the proliferative activity of immature elements. At the highest doses the proliferation area is almost completely absent and the elements still present are strongly degenerated. Acriflavine, at the concentration able to reduce or to inhibit the growth of control cultures, is ineffective in altering the ratio of immature elements in cultures of immunized animals. The ability of these elements to incorporate 3H thymidine is also unchanged.

The Vent-Like Homeobox Gene VENTX Promotes Human Myeloid Development and Is Highly Expressed In Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 739-739
Author(s):  
Vijay P. S. Rawat ◽  
Natalia Arseni ◽  
Farid Ahmed ◽  
Medhanie A. Mulaw ◽  
Silvia Thoene ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Myeloid Cells ◽  
Lymphoid Cells ◽  
Hematopoietic Development ◽  
Cd34 Cells ◽  
The Impact

Abstract Abstract 739 Recent studies suggest that a variety of regulatory molecules active in embryonic development such as clustered and non-clustered homeobox genes play an important role in normal and malignant hematopoiesis. Since it was shown that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, it is of particular interest to examine the expression profile and function of its only recently discovered human homologue VENTX in hematopoietic development. Expression of the VENTX gene was analyzed in normal human hematopoiesis and AML patients samples by microarray and qPCR. To test the impact of the constitutive expression of VENTX on human progenitor cells, CD34+ cord blood (CB) cells were retrovirally transduced with VENTX or the empty control vector and analyzed using in vitro and in vivo assays. So far we and others have not been able to identify a murine Xenopus xvent gene homologue. However, we were able to document the expression of this gene by qPCR in human lineage positive hematopoietic subpopulations. Amongst committed progenitors VENTX was significantly 13-fold higher expressed in CD33+ BM myeloid cells (4/4 positive) compared to CD19+ BM lymphoid cells (5/7 positive, p=0.01). Of note, expression of VENTX was negligible in normal CD34+/CD38− but detectable in CD34+ BM human progenitor cells. In contrast to this, leukemic CD34+/CD38− from AML patients (n=3) with translocation t(8,21) showed significantly elevated expression levels compared to normal CD34+ BM cells (n=5) (50-fold higher; p≤0.0001). Furthermore, patients with normal karyotype NPM1c+/FLT3-LM− (n=9), NPM1c−/FLT3-LM+ (n=8) or patients with t(8;21) (n=9) had an >100-fold higher expression of VENTX compared to normal CD34+ BM cells and a 5- to 7.8-fold higher expression compared to BM MNCs. Importantly, lentivirus-mediated long-term silencing of VENTX in human AML cell lines (mRNA knockdown between 58% and 75%) led to a significant, reduction in cell number compared to the non-silencing control construct (>79% after 120h). Suggesting that growth of human leukemic cell lines depends on VENTX expression in vitro. As we observed that VENTX is aberrantly expressed in leukemic CD34+ cells with negligible expression in normal counterparts, we assessed the impact of forced VENTX gene expression in normal CD34+ human progenitor cells on the transcription program. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development (CD11b, CD125, CD9,CD14 and M-CSF), and downregulates genes involved in early lymphoid development (IL-7, IL-9R, LEF1/TCF and C-JUN) and erythroid development such as EPOR, CD35 and CD36. We then tested whether enforced expression of VENTX in CD34+ cells is able to alter the hematopoietic development of early human progenitors as indicated by gene expression and pathway analyses. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells induced a significant increase in the number of myeloid colonies compared to the GFP control with 48 ± 6.5 compared to 28.9 ± 4.8 CFU-G per 1000 initially plated CD34+ cells (n=11; p=0.03) and complete block in erythroid colony formation with an 81% reduction of the number of BFU-E compared to the control (n=11; p<0.003). In a feeder dependent co-culture system, VENTX impaired the development of B-lymphoid cells. In the NOD/SCID xenograft model, VENTX expression in CD34+ CB cells promoted generation of myeloid cells with an over 5-fold and 2.5-fold increase in the proportion of human CD15+ and CD33+ primitive myeloid cells compared to the GFP control (n=5, p=0.01). Summary: Overexpression of VENTX perturbs normal hematopoietic development, promotes generation of myeloid cells and impairs generation of lymphoid cells in vitro and in vivo. Whereas VENTX depletion in human AML cell lines impaired their growth.Taken together, these data extend our insights into the function of human embryonic mesodermal factors in human hematopoiesis and indicate a role of VENTX in normal and malignant myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

Most Acute Myeloid Leukemia Progenitor Cells With Long-Term Proliferative Ability In Vitro and In Vivo Have the Phenotype CD34+/CD71−/HLA-DR−

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4325-4335 ◽  
Author(s):  
A. Blair ◽  
D.E. Hogge ◽  
H.J. Sutherland
Keyword(s):  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Scid Mice ◽  
Hla Dr ◽  
Proliferation In Vitro ◽  
Acute Myeloid

Acute myeloid leukemia (AML) occurs as the result of malignant transformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML cells are capable of proliferation in vitro, suggesting that AML cells may be organized in a hierarchy, with only the most primitive of these cells capable of maintaining the leukemic clone. To further investigate this hypothesis, we have evaluated a strategy for purifying these primitive cells based on surface antigen expression. As an in vitro endpoint, we have determined the phenotype of AML progenitor cells which are capable of producing AML colony-forming cells (CFU) for up to 8 weeks in suspension culture (SC) and compared the phenotype with that of cells which reproduce AML in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activated cell sorted (FACS) for coexpression of CD34 and CD71, CD38, and/or HLA-DR and the subfractions were assayed in vitro and in vivo at various cell doses to estimate purification. While the majority of primary AML CFU lacked expression of CD34, most cells capable of producing CFU after 2 to 8 weeks in SC were CD34+/CD71−. HLA-DR expression was heterogeneous on cells producing CFU after 2 to 4 weeks. However, after 6 to 8 weeks in SC, the majority of CFU were derived from CD34+/HLA-DR− cells. Similarly, the majority of cells capable of long-term CFU production from SC were CD34+/CD38−. Most cells that were capable of engrafting NOD/SCID mice were also CD34+/CD71− and CD34+/HLA-DR−. Engraftment was not achieved with CD34+/CD71+ or HLA-DR+subfractions, however, in two patients, both the CD34+and CD34− subfractions were capable of engrafting the NOD/SCID mice. A three-color sorting strategy combining these antigens allowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cells in one experiment. Phenotyping studies suggest even higher purification could be achieved by combining lack of CD38 expression with the CD34+/CD71− or CD34+/HLA DR− phenotype. These results suggest that most AML cells capable of long-term proliferation in vitro and in vivo share the CD34+/CD71−/HLA-DR− phenotype with normal stem cells. Our data suggests that in this group of patients the leukemic transformation has occurred in a primitive progenitor, as defined by phenotype, with some degree of subsequent differentiation as defined by functional assays.

scholarly journals Leptin Receptor As a Functional Marker for Long-Term Repopulating Hematopoietic Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3712-3712
Author(s):  
Thao Trinh ◽  
Scott Cooper ◽  
Arafat Aljoufi ◽  
Edward F. Srour ◽  
Hal E. Broxmeyer
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Functional Marker ◽  
Type I ◽  
Hematopoietic Stem ◽  
Lepr Expression ◽  
Cell Stem Cell

Hematopoietic cell transplantation is an invaluable life-saving regimen for patients affected by malignant and non-malignant hematological disorders. However, successful clinical outcomes depend on the abilities of hematopoietic stem (HSCs) and progenitor cells (HPCs) to home to the bone marrow (BM) and then reconstitute a healthy new blood system. Leptin (Lep), a metabolic hormone well-characterized for its regulations of appetite and body weight by acting on the hypothalamus neurons, has a WSXWS motif of the type I cytokine receptor family and has reported hematopoietic effects (Cioffi et al., Nat Med 1996, Bennett et al., Curr Biol 1996, Umemoto et al., Blood 1997, Gainsford et al. Proc Natl Acad Sci USA 1996, Claycombe et al., Proc Natl Acad Sci USA 2008). These studies were however mostly limited to in vitro assays. Recent work demonstrated that Lep receptor(r)+ stromal cells were indispensable for maintenance of HSC/HPC (Comazzetto et al., Cell Stem Cell 2019, Himburg et al., Cell Stem Cell 2018, Zhou et al., Nat Cell Biol 2017). Yet, whether Lepr expression on HSC/HPC has effects on their in vivo functions remain largely unknown. We hypothesized that environmental factors that affect metabolism of HSCs and HPCs, such as those modulated by Lep/Lepr interactions, may be involved in HSC/HPC regulation and the engraftment of these cells. Using flow cytometry analysis, we first assessed expression levels of Lepr on HSCs and HPCs. While only a low percentage of mouse BM HSC/HPC expressed Lepr, both the percentages of Lepr+HSCs (28.5% Lepr+LT-HSC and 17.2% Lepr+ST-HSC) and mean fluorescence intensity (MFI) of surface Lepr on these cells are significantly higher than that of Lepr+HPCs such as CMP, GMP and CLP (3.8%, 1.5%, 0.7% Lepr+ respectively). Despite the fact that HPCs express a lower level of Lepr, intact Lep/Lepr signaling was critical for their functions. This was illustrated by in vitro colony assay of cells taken from Lepr knockout (-/-) mouse BM in which significantly fewer absolute numbers per femur of HPC-derived colonies (CFU-GM, CFU-GEMM, BFU-E) formed compared to WT controls, and these progenitors were in a slow or non-cycling state. To evaluate how Lepr expression affects in vivo HSC/HPC functions, equal numbers of BM C57BL/6 (WT; CD45.2+) Lepr - Lineage-Sca1+cKit+ (LSK) vs. Lepr+LSK cells were sorted and each transplanted with competitive BoyJ (CD45.1+) cells into lethally irradiated CD45.2+/CD45.1+ F1 recipients. A consistently higher engraftment capacity of Lepr+LSK cells was manifested in comparison to Lepr - LSK cells as noted in peripheral blood (PB) at months 1-6 chimerism post-transplant (91% vs 1.1% at month 6). Lepr+HSCs and Lepr+MPPs expressed similar levels of surface CXCR4 in comparison to corresponding Lepr - populations, suggesting that homing differences may not explain increased engraftment of Lepr+ LSK. At month 6, Lepr+LSK, but not Lepr - cells, demonstrated a significant myeloid-biased engraftment (0.24 vs 0.03 respectively for myeloid/lymphoid ratios). This is consistent with the phenotypic finding that compared to Lepr -LSK cells, Lepr+LSK cells contained a significantly lowered percentage of MPP4 progenitor cells (3.6% vs 36%), which have been demonstrated as a lymphoid-biased subset of MPPs (Pietras et al., Cell Stem Cell 2015). In addition, Lepr+LSK cells contained three-fold fewer progenitors as determined by in vitro colony assays. These findings demonstrated that Lepr+LSK cells were enriched for long-term hematopoietic repopulating HSCs, while its counterpart Lepr -LSK cells contained mostly HPCs. The data also suggested that absence of Lepr expression may play a role in fate-decision skewing HSCs towards MPP4 production. For beginning efforts at mechanistic insight, we hypothesized that Lepr+ HSCs and Lepr+MPP may be different than Lepr - cells in mitochondrial activity. Compared to Lepr - cells, Lepr+HSC and Lepr+MPP cells interestingly possessed more robust mitochondrial metabolism, as demonstrated by their mitochondria having significantly higher membrane potential (measured by JC-1 assay). In summary, Lep/Lepr signaling appears to be a functional ligand-receptor axis for maintaining HSC/HPC homeostasis and differentiation cell bias. Moreover, Lepr expression may serve as a functional marker for long-term repopulating HSCs, which has potential translational possibilities, as Lepr is highly conserved between mice and humans. Disclosures No relevant conflicts of interest to declare.

Leptin Receptor As a Marker for a Subset of Long-Term Functional Hematopoietic Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Thao Trinh ◽  
James Ropa ◽  
Arafat Aljoufi ◽  
Scott Cooper ◽  
Edward F. Srour ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Leptin Receptor ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Type I ◽  
Hematopoietic Stem ◽  
Therapeutic Implications

The hematopoietic system is maintained by the hematopoetic stem and progenitor cells (HSCs/HPCs), a group of rare cells that reside in a hypoxic bone marrow (BM) microenvironment. Leptin (Lep) is well-known for its neuroendocrine and immunological functions, and its receptor (Lepr) has been studied extensively in the BM niche cells. Yet, its biological implications in HSC/HPC biology remained largely unknown. In this study, we hypothesized that Lepr-expressing HSCs/HPCs are functionally and transcriptomically distinct from their negative counterparts. To test our hypothesis, we utilized both in vitro and in vivo approaches. We first employed Fluorescence-activated cell sorting (FACS) analysis to confirm expression of Lepr on HSCs/HPCs in adult mouse BM. We then isolated equal numbers of Lepr+Lineage-Sca1+cKit+ (LSK cells - a heterogenous population of long-term, short-term HSCs and multipotent HPCs) and Lepr-LSK cells from C57BL/6 (CD45.2+) mouse BM to perform colony-forming unit (CFU) assay and competitive transplantation assay, which also included using competitor cells from BoyJ (CD45.1+) unseparated BM and lethally-irradiated F1 (CD45.1+CD45.2+) as hosts. To determine whether Lepr can further hierarchize HSCs into two distinct populations, we repeated the competitive transplants using freshly isolated C57BL/6 Lepr+HSCs or Lepr-HSCs cells instead. At the end of primary transplants, whole BM were analyzed for donor chimerisms in the peripheral blood (PB) and BM as well as transplanted in a non-competitive fashion into lethally-irradiated secondary recipients. To gain mechanistic insights, we assessed homing potential as homing plays a role in increased engraftment. We also performed bulk RNA-seq using freshly sorted BM Lepr+HSCs or Lepr-HSCs to elucidate potential molecular pathways that are responsible for the differences in their functional capacity. By phenotypic studies, our FACS analyses showed that Lepr+ cells represented a smaller population within the hematopoietic compartment in the BM. However, HSCs contained a higher percentage of Lepr+ cells than other HPC populations. By functional assessments, Lepr+LSK cells were more highly enriched for colony-forming progenitor cells in CFU assay as compared to Lepr-LSK cells. Interestingly, Lepr+LSK cells exhibited more robust engraftment capability in primary transplants and substantial self-renewal capacity in secondary transplants throughout different time points in both PB and BM. In addition, Lepr+HSCs showed significantly higher donor chimerisms in PB month 1, 2, 4 and BM month 4 with similar lineage output compared to Lepr-HSCs. Higher engraftment could be due to increased homing of HSCs to the BM; however, Lepr+HSCs and Lepr-HSCs showed similar homing capacity as well as levels of surface CXCR4 expression. Molecularly, Fast Preranked Gene Set Enrichment Analysis (FGSEA) showed that Lepr+HSCs were enriched for Type-I Interferon and Interferon-gamma response pathways with Normalized Enrichment Scores of 2 or higher. Lepr+HSC transcriptomic study also revealed that these cells as compared to Lepr-HSCs expressed significantly higher levels of genes involved in megakaryopoiesis and proinflammatory immune responses including the NF-κB subunits (Rel and Relb). Interestingly, both IFN-γ and NF-κB signalings have been demonstrated to be critical for the emergence of HSCs from the hemogentic endothelium during embryonic development. In summary, although Lepr+LSK cells occupied a minor fraction compared to their negative counterparts in the BM, they possessed higher colony-forming capacity and were more highly enriched for long-term functional HSCs. In line with this, Lepr+HSCs engrafted significantly higher and self-renewed more extensively than Lepr-HSCs, suggesting that Lepr not only can be used as a marker for functional HSCs but also further differentiate HSCs into two functionally distinguishable populations. Intriguingly, Lepr+HSCs were characterized with a proinflammatory transcriptomic profile that was previously suggested to be critical for the development of HSCs in the embryo. All together, our work demonstrated that Lepr+HSCs represent a subset of highly engrafting adult BM HSCs with an embryonic-like transcriptomic signature. This can have potential therapeutic implications in the field of hematopoietic transplantation as Lepr is highly conserved between mice and human. Disclosures No relevant conflicts of interest to declare.

Coagulation Factor Thrombin Regulates Hematopoietic Stem and Progenitor Cell Egress and Mobilization Via PAR-1 & CXCR4 Upregulation, SDF-1 Secretion and EPCR Shedding

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2341-2341 ◽  
Author(s):  
Shiri Gur-Cohen ◽  
Tomer Itkin ◽  
Aya Ludin ◽  
Orit Kollet ◽  
Karin Golan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Progenitor Cell ◽  
Single Injection ◽  
Hematopoietic Stem ◽  
Epcr Shedding

Abstract Abstract 2341 Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions. The G-protein-coupled receptor protease-activated receptor-1 (PAR-1) and its activator thrombin play an important role in coagulation following injury and bleeding. We report that a single injection of thrombin induced rapid HSPC mobilization within one hour, increasing circulating leukocytes, predominantly CFU-C and primitive Lin−/Sca-1+/c-Kit+ (SKL) progenitor cells. This rapid mobilization was preceded by a dramatic decrease of SDF-1 (CXCL12) in BM stromal cells, including rare Nestin+ mesenchymal stem cells (MSC) which functionally express PAR-1 and release SDF-1. Thrombin injection also increased expression of PAR-1 and CXCR4 by BM HSPC. These results suggest involvement of the coagulation cascade of thrombin & PAR-1 in rapid SDF-1 secretion from niche supporting BM stromal cells as part of host defense and repair mechanisms. Administration of a PAR-1 specific antagonist (SCH79797) upregulated BM SDF-1 levels and significantly reduced the amounts of circulating CFU-C and primitive SKL progenitor cells. In vitro stimulation of BM mononuclear cells with thrombin for 1 hour led to increased CXCR4 expression by Lin−/c-Kit+ progenitors, accompanied by enhanced spontaneous and SDF-1 induced migration. Of note, specific PAR-1 inhibition in vitro significantly reduced SDF-1-directed migration of Lin-/c-Kit+ progenitors. Mechanistically, we found that thrombin - activated PAR-1 induced the downstream p38 MAPK and eNOS (nitric oxide synthase) signaling pathways. Long term repopulating hematopoietic stem cells (HSC) in murine BM highly express endothelial protein C receptor (EPCRhigh) (Balazs & Mulligan et al Blood 2006; Kent & Eaves et al Blood 2009). EPCR is expressed primarily on endothelial cells (EC) and has anti coagulation and anti inflammatory roles. Surface EPCR expression on EC is downregulated by many factors, including PAR-1 activation by thrombin, a process which is termed shedding and is not fully understood. Importantly, we found that over 90% of BM CD45+/EPCRhigh long-term HSC express PAR-1 and that circulating primitive HSPC in the blood and spleen lack EPCRhigh expression. In addition, in-vivo thrombin administration downregulated EPCR from BM HSC via eNOS signaling, thus allowing the release of stem cells from their BM microenvironment anchorage to the circulation. Correspondingly, in eNOS deficient mice, thrombin failed to induce PAR-1 upregulation, EPCR shedding, and HSPC mobilization. Recently, we reported that the antioxidant NAC inhibits G-CSF induced mobilization (Tesio & Lapidot et al Blood 2011). Co-administration of G-CSF with NAC prevented PAR-1 upregulation, concomitantly with reduced HSPC mobilization and increased levels of EPCRhigh HSC in the BM. Treatment of PAR-1 antagonist with G-CSF inhibited PAR-1 and CXCR4 upregulation on BM leukocytes and immature Lin−/c-Kit+ cells accompanied by increased levels of BM EPCRhigh HSC and reduced HSPC mobilization. Tissue factor (TF) is the main initiator of the coagulation system via the formation of an enzymatic “prothrombinase complex” that converts prothrombin to active thrombin. Unexpectedly, we found a unique structure of cell clusters expressing TF, located preferentially in the trabecular-rich area of the femoral metaphysis in murine bone tips, a region highly exposed to osteoclast/osteoblast bone remodeling. In vitro, immature osteoclasts exhibited increased TF expression in cell fusion areas, suggesting that in vivo osteoclast maturation activates the coagulation thrombin/PAR-1 axis of HSPC migration to the circulation. Finally, mimicking bacterial infection a single injection of Lipopolysaccharide (LPS), rapidly and systemically upregulated TF in the murine BM. LPS treatment prompted an increase in thrombin generation and subsequently HSPC mobilization, which was blocked by the PAR-1 antagonist. In conclusion, our study reveals a new role for the coagulation signaling axis, which acts on both hematopoietic and stromal BM cells to regulate steady state HSPC egress and enhanced mobilization from the BM. This thrombin/PAR-1 signaling cascade involves SDF-1/CXCR4 interactions, immature osteoclast TF activity, Nestin+/PAR-1+ MSC secretion of SDF-1 and EPCR shedding from hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Most Acute Myeloid Leukemia Progenitor Cells With Long-Term Proliferative Ability In Vitro and In Vivo Have the Phenotype CD34+/CD71−/HLA-DR−

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4325-4335 ◽  
Author(s):  
A. Blair ◽  
D.E. Hogge ◽  
H.J. Sutherland
Keyword(s):  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Scid Mice ◽  
Hla Dr ◽  
Proliferation In Vitro ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) occurs as the result of malignant transformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML cells are capable of proliferation in vitro, suggesting that AML cells may be organized in a hierarchy, with only the most primitive of these cells capable of maintaining the leukemic clone. To further investigate this hypothesis, we have evaluated a strategy for purifying these primitive cells based on surface antigen expression. As an in vitro endpoint, we have determined the phenotype of AML progenitor cells which are capable of producing AML colony-forming cells (CFU) for up to 8 weeks in suspension culture (SC) and compared the phenotype with that of cells which reproduce AML in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activated cell sorted (FACS) for coexpression of CD34 and CD71, CD38, and/or HLA-DR and the subfractions were assayed in vitro and in vivo at various cell doses to estimate purification. While the majority of primary AML CFU lacked expression of CD34, most cells capable of producing CFU after 2 to 8 weeks in SC were CD34+/CD71−. HLA-DR expression was heterogeneous on cells producing CFU after 2 to 4 weeks. However, after 6 to 8 weeks in SC, the majority of CFU were derived from CD34+/HLA-DR− cells. Similarly, the majority of cells capable of long-term CFU production from SC were CD34+/CD38−. Most cells that were capable of engrafting NOD/SCID mice were also CD34+/CD71− and CD34+/HLA-DR−. Engraftment was not achieved with CD34+/CD71+ or HLA-DR+subfractions, however, in two patients, both the CD34+and CD34− subfractions were capable of engrafting the NOD/SCID mice. A three-color sorting strategy combining these antigens allowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cells in one experiment. Phenotyping studies suggest even higher purification could be achieved by combining lack of CD38 expression with the CD34+/CD71− or CD34+/HLA DR− phenotype. These results suggest that most AML cells capable of long-term proliferation in vitro and in vivo share the CD34+/CD71−/HLA-DR− phenotype with normal stem cells. Our data suggests that in this group of patients the leukemic transformation has occurred in a primitive progenitor, as defined by phenotype, with some degree of subsequent differentiation as defined by functional assays.

scholarly journals Long-Term Transgene Expression in Proliferating Cells Mediated by Episomally Maintained High-Capacity Adenovirus Vectors

2004 ◽  
Vol 78 (1) ◽  
pp. 9-22 ◽  
Author(s):  
Florian Kreppel ◽  
Stefan Kochanek
Keyword(s):  
Transgene Expression ◽  
High Capacity ◽  
Target Cells ◽  
Resting Cells ◽  
Proliferating Cells ◽  
Daughter Cells ◽  
Adenovirus Vectors

ABSTRACT High-capacity “gutless” adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.

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