Efficiency of Homing and Engraftment Is Higher in VEGFR-3+CD34+CD38- Cells Than in VEGFR-3-CD34+CD38- Cells in Leukemic Patients

Abstract Surviving leukemic stem cells (LSCs) after chemotherapy lead to relapses in acute myeloid leukemia (AML). Because LSCs will not be eradicated after standard chemotherapy, inhibiting the propagation of AML cells by LSCs is a major part in AML treatment. Although CD34+CD38- cells in leukemia are representative LSCs, it is still difficult to distinguish them apart from normal hematopoietic stem cells (HSCs). So far, many studies have shown rapid advances in phenotypic characterization. However, heterogeneous diversity in AML patients may not allow effective eradication of LSCs. Vascular endothelial growth factor receptors (VEGFR)-3 is expressed in AML blasts and in the bone marrow (BM) environment including sinusoidal vessels. In particular, VEGFR-3 is strongly correlated with poor prognosis, leukemic cell proliferation and survival in AML. Based on previous reports, we were able to hypothesize that VEGFR-3 is expressed in LSCs and functions as a LSC marker. Here, we observed high expressions of VEGFR-3 on CD34+CD38− cells in AML patients who received chemotherapy and further showed low homing and engraftment capacity of CD45dim blast cells in the BM by a VEGFR-3 antagonist. In order to determine the expression of VEGFR-3 on LSCs, data was collected from 64 AML patients, 12 of whom were after complete remission (CR), as well as from 14 healthy volunteers. MNCs were first isolated and were then subjected to FACS analysis and immunocytochemistry. NOD-Scid IL2RγNull mice were used for homing efficiency and engraftment in vivo. MAZ51 was used as a VEGFR-3 antagonist. FACS analysis showed that VEGFR-3 was increased on CD34+CD38− LSC cells. (Normal vs. AML vs. CR, VEGFR-3 on CD34+CD38- cells: 8.33 ± 4.37% vs. 25.62 ± 2.46% vs. 23.46 ± 5.47%, P < 0.05). Similarly, immunocytochemistry clearly displayed the co-expression of VEGFR-3 on isolated CD34+CD38− LSC cells, suggesting the possibility of it as a LSC marker. We checked the ability of LSCs to use colony forming units assay. VEGFR-3+CD34+ cells showed unarguably enhanced colony forming ability compared to that of VEGFR-3-CD34+ cells from patients. To test whether VEGFR-3-CD34+ cells are not on apoptotic procedure, annexin-V and proliferation assay with Ki67 were performed, and there was no difference in apoptotic and proliferative movement in both cells. Intending to determine homing efficiency and engraftment, CD34 and CD45 markers were used in the BM and it was discovered that sorted VEGFR-3+CD34+CD38- cells showed significantly increased homing and engraftment efficiency compared to those of VEGFR-3-CD34+CD38- cells (by FACS, homing capacity: 1.93 ± 0.19% vs. 0.13 ± 0.06%, P = 0.02; engraftment: 26.4 ± 9.42% vs. 5.30 ± 2.31%, P < 0.05), implying that VEGFR-3 can serve as a marker for LSCs. We demonstrated that VEGFR-3 was highly expressed and enriched on CD34+CD38- cells in CR status as well as in the initial diagnosis of AML. Therefore, the targeting of VEGFR-3 may diminish LSC function in human AML. These findings could suggest some clues to develop therapeutic strategies targeting VEGFR-3+ LSCs with favorable tumor microenvironments. Disclosures No relevant conflicts of interest to declare.

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T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Blood ◽

10.1182/blood.v114.22.3980.3980 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3980-3980 ◽

Cited By ~ 1

Author(s):

Claudia Oancea ◽

Brigitte Rüster ◽

Jessica Roos ◽

Afsar Ali Mian ◽

Tatjana Micheilis ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Population ◽

Conflicts Of Interest ◽

Cell Model ◽

Leukemic Cell ◽

Hematopoietic Stem ◽

Proliferation Potential

Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

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Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽

10.1182/blood.v95.9.2813.009k20_2813_2820 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2813-2820 ◽

Cited By ~ 215

Author(s):

Lisa Gallacher ◽

Barbara Murdoch ◽

Dongmei M. Wu ◽

Francis N. Karanu ◽

Mike Keeney ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Surface Markers ◽

Cell Surface Markers ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

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High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Blood ◽

10.1182/blood-2002-03-0767 ◽

2003 ◽

Vol 101 (5) ◽

pp. 1759-1768 ◽

Cited By ~ 110

Author(s):

Bernhard Schiedlmeier ◽

Hannes Klump ◽

Elke Will ◽

Gökhan Arman-Kalcek ◽

Zhixiong Li ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Therapeutic Window ◽

Hematopoietic Stem ◽

Expression Levels ◽

Cd34 Cells ◽

Cord Blood Cd34 ◽

The Impact

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P = .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

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Ex-Vivo Expansion of Multipotent CD133+ Stem Cells with VEGF, FLT3l and SCGF.

Blood ◽

10.1182/blood.v104.11.4147.4147 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4147-4147

Author(s):

Sonja Loges ◽

Martin Butzal ◽

Uta Fischer ◽

Ursula M. Gehling ◽

Dieter K. Hossfeld ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Stem Cell ◽

Culture System ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Facs Analysis ◽

Hematopoietic Stem ◽

Large Numbers

Abstract The rare CD133+ stem cell population contains both hematopoietic and endothelial progenitors. Successful ex-vivo expansion of this multipotent population would therefore be of great benefit in many clinical settings including stem cell transplantation and gene therapy. We developed a cell culture system containing the recombinant human cytokines vascular endothelial growth factor (VEGF), FLT3 ligand (FLT3L) and stem cell growth factor (SCGF) for ex-vivo expansion of purified human CD133+ stem cells obtained from leukapheresis products from patients pre-treated with G-CSF. FACS analysis, colony assays and NOD-SCID transplantation studies were performed to monitor stem cell and endothelial phenotype in-vitro and in-vivo. Cultivation with VEGF, FLT3L and SCGF induced a mean 2200-fold increase of total cell counts in 5 weeks. FACS analysis revealed persistence of 6–15% CD133+ stem cells indicating proliferation and survival of primitive hematopoietic stem cells. 5–6% of the proliferating cells expressed the endothelial markers CD144 (VE-Cadherin) and von-Willebrand factor (vWF). Ex-vivo expanded stem cells could be differentiated into adherent endothelial cells after withdrawal of SCGF and FLT3L allowing generation of large numbers of endothelial cells. Colony-assays showed an increase of hematopoietic and endothelial colonies after 5 weeks of ex-vivo expansion indicating simultaneous proliferation of hematopoietic and endothelial precursors under the established culture conditions (CFU-E 60-fold, CFU-GEMM 48-fold, CFU-GM 59-fold, CFU-G 99-fold, CFU-M 1356-fold and CFU-EC 1843-fold). To assess in-vivo functionality, hematopoietic stem cells expanded ex-vivo for 7, 14, 21 and 32 days were transplanted into sublethally irradiated NOD-SCID mice. For each expansion period, the mean percentage of anti-human CD45 positive bone marrow cells 3 months post-transplantation was 11, 3, 3 and 1%, respectively. Human CD45+ cells for each set of experiments contained a mean of 15, 26, 8 and 32% T-cells (CD3+), 9, 0, 7 and 21% B-cells (CD19+), 24, 2, 2 and 11% monocytes (CD14+), 21, 3, 1 and 12% granulocytes (CD33+) and 19, 37, 44 and 24% stem cells (CD34+) (d7 (n=5), d14 (n=4), d21 (n=7) and d32 (n=6) respectively). Our experiments showed multilineage engraftment of human stem cells expanded for more than 4 weeks ex-vivo. Therefore our culture system provides a tool to generate large numbers of human stem and endothelial cells for clinical purposes.

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Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽

10.1182/blood.v104.11.2685.2685 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2685-2685

Author(s):

A. Daisy Narayan ◽

Jessica L. Chase ◽

Adel Ersek ◽

James A. Thomson ◽

Rachel L. Lewis ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Blood Samples ◽

Human Dna ◽

Cd34 Cells ◽

Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

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Xenotransplantation of Primary CD34+CD38- Hematopoietic Stem Cells Results in an In Vivo Model of Idiopathic Myelofibrosis.

Blood ◽

10.1182/blood.v108.11.666.666 ◽

2006 ◽

Vol 108 (11) ◽

pp. 666-666

Author(s):

Noriyuki Saito ◽

Fumihiko Ishikawa ◽

Kazuya Shimoda ◽

Shuro Yoshida ◽

Yoriko Saito ◽

...

Keyword(s):

Stem Cells ◽

In Vivo Model ◽

Idiopathic Myelofibrosis ◽

Hematopoietic Stem ◽

Hematopoietic Reconstitution ◽

Cd34 Cells ◽

Normal Human ◽

Myelomonocytic Cells ◽

Xenotransplantation Model

Abstract Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These abnormal cells secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and lead to extramedullary hematopoiesis and the appearance of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate at the level of hematopoietic stem cell (HSC), this has not been demonstrated directly in primary human IMF. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we used the newborn NOD/SCID/IL2rg-null xenotransplantation model. We purified PB CD34+ cells from six IMF patients, transplanted 1–10 x10e4 cells intravenously into newborn NOD/SCID/IL2rg-null recipients and analyzed PB and BM human CD45+ hematopoietic cell chimerism, degree of suppression of murine hematopoiesis, presence of hallmark BM fibrosis and plasma TGF-b1 levels in the recipients at 6 months post-transplantation. Primary IMF PB CD34+ cells from five out of six patients engrafted in twelve out of twelve recipients. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of murine fibroblasts, the presence of human megakaryocytes and elevated plasma TGF-b1 levels, recapitulating the clinical features of IMF. Three distinct patterns of human hematopoietic reconstitution were observed among the engrafted recipients: Predominantly malignant myelomonocytic engraftment in the PB and BM (n=4), Reconstitution of both normal human hematopoiesis (with mature B and T cells, myeloid cells and platelets) and malignant myelomonocytic cells (n=6) and Development of acute leukemia (n=2). Fibrotic change was seen even in the BM of recipients that showed normal human hematopoietic reconstitution, showing that in IMF, there is co-existence of both normal and malignant hematopoietic stem/progenitor cells in the PB CD34+ fraction. Furthermore, when 5–10 x 10e3 sorted PB CD34+CD38– cells from three patients were transplanted into six newborn NOD/SCID/IL2rg-null recipients, reconstitution with human myelomonocytic cells associated with BM fibrosis was demonstrated in all recipients, with compatible level of PB and BM chimerism with those transplanted with PB CD34+ cells. These findings demonstrate that the IMF-initiating cells are contained within the HSC fraction. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and may be useful for development of novel therapeutic agents for IMF.

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LYL1, Class II Basic Helix-Loop-Helix Transcription Factor, Induces the Differentiation of Common Marmoset Embryonic Stem Cells to Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2348.2348 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2348-2348

Author(s):

Hirotaka Kawano ◽

Tomotoshi Marumoto ◽

Michiyo Okada ◽

Tomoko Inoue ◽

Takenobu Nii ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Lymphoblastic Leukemia ◽

Embryonic Stem ◽

Common Marmoset ◽

Hematopoietic Stem ◽

Helix Loop Helix ◽

Cd34 Cells

Abstract Abstract 2348 Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors. The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset. To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34+ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1. Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias. Disclosures: No relevant conflicts of interest to declare.

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Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽

10.1182/blood.v120.21.1224.1224 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1224-1224

Author(s):

Junke Zheng ◽

Chengcheng Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Actin Binding ◽

Wild Type ◽

Cell Fates ◽

Hematopoietic Stem ◽

Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Induced Hematopoietic Differentiation Of Common Marmoset Embryonic Stem Cells By LYL1 Can Give Rise To Hematopoietic Stem Cells Having The Enhanced Bone Marrow Reconstitution Capability

Blood ◽

10.1182/blood.v122.21.1192.1192 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1192-1192

Author(s):

Hirotaka Kawano ◽

Tomotoshi Marumoto ◽

Takafumi Hiramoto ◽

Michiyo Okada ◽

Tomoko Inoue ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Lymphoblastic Leukemia ◽

Embryonic Stem ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Hsc Transplantation

Abstract Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host’s hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro. The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point. Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures: No relevant conflicts of interest to declare.

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Inhibition Of CXCR2 As a Therapeutic Strategy In AML and MDS

Blood ◽

10.1182/blood.v122.21.484.484 ◽

2013 ◽

Vol 122 (21) ◽

pp. 484-484 ◽

Cited By ~ 1

Author(s):

Carolina Schinke ◽

Orsolya Giricz ◽

Shanisha A. K. Gordon ◽

Laura Barreyro ◽

Tushar D. Bhagat ◽

...

Keyword(s):

Stem Cells ◽

Leukemic Cell ◽

Leukemia Stem Cells ◽

P Value ◽

Healthy Controls ◽

Functional Studies ◽

Cxcr2 Expression ◽

Cd34 Cells

Abstract Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS) arise from accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and/or committed progenitors. A series of transforming events can initially give rise to pre-leukemia stem cells (pre-LSC) as well as fully transformed leukemia stem cells (LSC), both of which need to be targeted in strategies aimed at curing these diseases. We conducted parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations in individual patients of MDS and AML (N=16) and identified candidate genes that are consistently dysregulated at multiple immature stem and progenitor cell stages. Interleukin 8 (IL8), was one of the most consistently overexpressed genes in MDS/AML Hematolpoetic Stem Cells (HSCs) and progenitors when compared to healthy control HSCs and progenitors. IL8 is a pro-inflammatory chemokine, which is able to activate multiple intracellular signaling pathways after binding to its surface receptor CXCR2. Even though increased IL8-CXCR2 signaling has been shown to promote angiogenesis, metastasis and chemotherapy resistance in many solid tumors, its role in AML and MDS is not well elucidated. We further analyzed gene expression profiles of CD34+ cells from 183 MDS patients and found significant increased expression of CXCR2 in MDS when compared to healthy controls (FDR<0.1). Most importantly, analysis of The Cancer Genome Atlas (TCGA) AML (n=200) dataset showed that CXCR2 expression was predictive of significantly adverse prognosis (log rank P value=0.0182; median survival of 245 days in cxcr2 high vs 607 days in cxcr2 low) in patients, further pointing to a critical role of IL8-CXCR2 signaling in AML/MDS. Next, we studied the functional role of IL8 and CXCR2 in AML. A panel of leukemic cell lines (THP-1, U937, KG-1, MOLM13, HL-60, K532) were screened for CXCR2 expression and revealed significantly higher expression when compared to healthy CD34+ control cells. SB-332235, a specific inhibitor of CXCR2 was used for functional studies. CXCR2 inhibition led to significant, (p<0.05) reduction in proliferation in all 6 cell lines tested and an effect was seen as early as 24 hrs of exposure. CXCR2 inhibition was found to lead to G0/G1 cell cycle arrest and trigged apoptosis in THP-1 and U937 cells (p-value 0.004 and 0.02 respectively). Incubation of primary AML/MDS bone marrow samples with SB-332235 similarly lead to significantly reduced proliferation at 24hrs, when compared to healthy CD34+ cells. Selective, and highly significant inhibition of leukemic cell growth was also seen in colony assays from primary MDS/AML samples (mean leukemic colonies in AML/MDS= 73 vs 313 in controls, P < 0.001). Interestingly, inhibition of CXCR2 in primary AML marrow samples led to induction of apoptosis in immature CD34+/CD38- cells when compared to healthy controls. Lastly, xenografting studies with THP-1 leukemic cells revealed that CXCR2 inhibitor treatment led to decreased leukemic burden and organ infiltration when compared to placebo controls in vivo. In summary we have found significantly increased expression of IL8 and its receptor CXCR2 in sorted HSCs and progenitors from AML and MDS patients. High CXCR2 expression was a marker of adverse prognosis in a large cohort of AML patients. Most importantly, in vitro and in vivo functional studies showed that CXCR2 is a potential therapeutic target in AML/MDS and is able to selectively target immature, LSC-enriched cell fractions in AML. Disclosures: No relevant conflicts of interest to declare.

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