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

Blood ◽  
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.

Transplantation of Primary Human CD34+CD38 - Hematopoietic Stem Cells Recapitulates Idiopathic Myelofibrosis in the NOD/scid/IL2rgKO Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 260-260
Author(s):  
Noriyuki Saito ◽  
Fumihiko Ishikawa ◽  
Kazuya Shimoda ◽  
Shuro Yoshida ◽  
Yoriko Saito ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extramedullary Hematopoiesis ◽  
In Vivo Model ◽  
Fish Analysis ◽  
Human Stem Cells ◽  
Idiopathic Myelofibrosis ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Xenotransplantation Model

Abstract Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM leads to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38- cells from six IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Six out of thirteen recipients transplanted with patient HSCs exhibited human hematopoietic engraftment, and CD33+ myeloid cells accounted for 80.5+/−9.41% of all the engrafted CD45 + population (as compared with the recipients transplanted with normal HSCs). BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was < 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. To investigate the origin of BM fibroblast, we performed FISH analysis using human and mouse centromeric probes and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of sixty fibroblasts examined, fifty-four cells were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38- HSC fraction and these cells possess differentiation capacity to fibroblasts. 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 the development of novel therapeutic agents for IMF.

Analysis of Idiopathic Myelofibrosis Initiating Cell in NOD/SCID/IL2rgKO Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3715-3715
Author(s):  
Noriyuki Saito ◽  
Fumihiko Ishikawa ◽  
Kazuya Shimoda ◽  
Shuro Yoshida ◽  
Yoriko Saito ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extramedullary Hematopoiesis ◽  
In Vivo Model ◽  
Fish Analysis ◽  
Human Stem Cells ◽  
Idiopathic Myelofibrosis ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Xenotransplantation Model

Abstract Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM lead to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38− cells from four IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Twelve out of nineteen recipients transplanted with patient CD34+ cells or CD34+CD38− cells exhibited human hematopoietic engraftment, and the frequency of CD33+ myeloid cells (82.5+/−12.2% among the engrafted CD45+ cells) was higher than that in the recipients transplanted with normal HSCs. These CD33+ cells expressed other myelo-monocytic markers such as CD14, CD11b, CD15, and HLA-DR. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was < 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. With FISH analysis using human X probe and immuno-staining using anti-TGF-beta1 antibody, we could confirm the TGF-beta1 production of human megakaryocytes in the recipient BM. To investigate the origin of BM fibroblasts, we performed FISH analysis using human X chromosome probe and mouse centromeric probe, and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of one hundred fifty fibroblasts examined, one hundred thirty six cells (90.7%) were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38− HSC fraction and these cells possess differentiation capacity to fibroblasts. 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 the development of novel therapeutic agents for IMF.

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

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
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.

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

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1759-1768 ◽  
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.

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

Blood ◽  
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.

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

Blood ◽  
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.

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 ◽  
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.

The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 516-516 ◽  
Author(s):  
Daniel Goff ◽  
Alice Shih ◽  
Angela Court Recart ◽  
Larisa Balaian ◽  
Ryan Chuang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Blast Crisis ◽  
Chemotherapy Resistance ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4782-4782
Author(s):  
Heeje Kim ◽  
Ji-Yoon Lee ◽  
Sohye Park ◽  
Jae-Ho Yoon ◽  
Woo-Sung Min ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Leukemic Cell ◽  
Phenotypic Characterization ◽  
Facs Analysis ◽  
Hematopoietic Stem ◽  
Tumor Microenvironments ◽  
Cd34 Cells

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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