Differential Expression of SLAM Family Receptor Markers in Normal Human Hematopoietic Stem Cells and Their Malignant Counterpart in MDS and AML.

Abstract Members of the signaling lymphocytic activation molecule (SLAM) family, including CD150, CD48 and CD244, were shown to precisely distinguish more committed lineage restricted progenitor cells from pluripotent and multipotent murine hematopoietic stem cells (HSC; Kiel et al; 2005 Cell). Similar SLAM profiles may also be present on HSC subsets in humans. We hypothesized that these SLAM markers may be indicators not only of stem cell potential in normal hematopoiesis but also distinguish a subset of the most immature malignant precursors of leukemia. In agreement with the concept of a “cancer stem cell,” the presence of leukemic stem cell population may be an indicator of important clinical and biological properties. We first tested the distribution of CD150, CD48 and CD244 antigens on human CD34+ cells derived from 7 control individuals using 4-color flow cytometry. CD34+ cells were measured in the blast gate based on side scatter and CD45 expression. Within CD34+ blasts, expression of CD48, CD150, and CD244 was detected on 16.71%±9.69, 6.53%±2.93, and 26.92%±6.95 of cells respectively. Subsequently, we investigated SLAM expression in 9 immature leukemic cell lines, including KG-1, K562, U937, HEL, HL60, MKN-95, NB-4, Kasumi and UT7, and found increased expression of SLAM markers in KG-1 (CD48+, CD150+, CD244+) and Kasumi (CD48−, CD150−, CD244+). Consequently, none of the leukemic cells showed pluripotent/multipotent SLAM profiles. We then compared the SLAM marker expression on blasts from patients with AML and MDS with that of CD34+ cells from normal controls. We studied a total of 28 patients: 11 MDS (2 low grade, 5 advanced MDS, 3 MDS/MPD overlap) and 10 AML (FAB: 3 M1, 2 M2, 1 M3, 2 M4/M4E0 and 2 M6). In our cohort, 8/10 AML patients expressed one of the three SLAM markers; 6/10 were CD150−CD48−CD244+ (63.57%±6.96) and 2/10 were CD150+CD48−CD244−(46%±10.96) suggestive of the presence of either pluripotent or multipotent leukemic stem cell phenotype. In the MDS cohort, 8/11 patients expressed one of three SLAM markers, 7/11 were CD150−CD48−CD244+ (41.21% ± 8.9) and 1/11 were CD150+CD48−CD244− (1.26%±0.59) again consistent with a profile derived from either pluripotent or multipotent stem cells. None of the MDS and AML patients had either co-expression of CD244 and CD48 or increased expression of CD48 alone. Two of the M1 type AML patients with CD150−CD48−CD244+ phenotype received prior chemotherapy and achieved complete remission on bone marrow biopsy and flow cytometry using traditional blast markers. In some, we conclude that the SLAM receptor markers may be associated with certain types of leukemic blasts and may be useful in the identification of leukemic stem cell population in both MDS and AML.

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Granulocyte-Macrophage Progenitors (GMPs) Express Low Adhesive Potential and High CXCR-4 Levels

Blood ◽

10.1182/blood.v122.21.3698.3698 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3698-3698

Author(s):

Valentina Giai ◽

Christian Scharenberg ◽

Robert Wallin ◽

Florian Salomons ◽

Olaf Bergmann ◽

...

Keyword(s):

Stem Cell ◽

Cell Population ◽

The Other ◽

Matrix Proteins ◽

Stem Cell Population ◽

Adherent Cells ◽

Leukemic Stem Cell ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Cd34 Cells

Abstract Hematopoietic stem and progenitor cells (HSPCs) reside in a particular and specialized microenvironmental niche. Interaction of HSPCs with their niche is fundamental for cell proliferation, differentiation and quiescence. While alterations of HSPC-niche interactions have been shown to play an important role in animal models, methods to study the human HSPC niche are lacking. In order to characterize the adhesion of various HSPC subsets to various extracellular matrix proteins found in the bone marrow, we used high-throughput fluorescent microscopy to study cell adhesion by quantifying the ratio of adherent versus non-adherent cells being detached by gravity. This allowed the analysis of low cell numbers making the study of clinical samples feasible. Multiparameter flow cytometry was used to analyze both the adherent and non-adherent fractions to study the major subsets of CD34+ cells, evaluating CD34, CD38, CD45RA, CD123 and CD90 surface levels. More than 80% of plated CD34+ cells adhered to fibronectin, 70% to VCAM-1, while only a minor proportion adhered to laminin, osteopontin and collagen-1 (11%-19%). Inhibitory antibodies against α4 and α5 integrin were used to inhibit adhesion of CD34+ cells to fibronectin and VCAM-1. α4 integrin antibody significantly decrease the number of adherent CD34+ cells to fibronectin from 65% to 38% (p< 0.0001) and on VCAM-1 from 51% to 12% (p< 0.0001). No effect was obtained by α5 integrin antibody. When comparing the major subsets of CD34+ cells, megakaryocyte-erythroid progenitors (MEPs) were the subpopulation that adhered strongest to each substrate followed by common myeloid progenitors (CMPs) and hematopoietic stem cells (HSCs). By contrast, granulocyte-macrophage progenitors (GMPs) adhered less to each of the studied matrix proteins (Fig. 1a-1b). Expression levels of CD34 were higher in adherent than in non-adherent cells, while CD45RA+ cells showed the opposite pattern (Fig. 1c-1d). These data suggest that the level of differentiation influences the adhesion properties and that more immature cells have a higher affinity to niche elements. We then analyzed the expression of adhesion markers in the different CD34+ cells subsets. Unexpectedly, GMPs, the least adhesive population, showed higher expression of CXCR4, a key chemokine receptor involved in HSPCs’ homing and differentiation. GMPs have been described to constitute the leukemic stem cell population in AML. GMPs also showed a high expression of CD44, a glycoprotein required for migration, cell interactions and adhesion. HSCs showed higher levels of α2 integrin while MEPs expressed more α4 integrin. The other subpopulations, common myeloid progenitors (CMPs), multipotent progenitors (MPPs) and lymphoid-primed multipotent progenitors (LMPPs) showed lower levels of all studied adhesion markers. In conclusion, we present a method to quantify the adhesion of different human HPSC subpopulations. The assay is suitable for analysis of human myeloid neoplasms and may elucidate aberrant patterns as well as reveal potential targets for anti-adhesive therapy inhibiting the niche«s ability to sustain the leukemic stem cell population. MEPs show more adhesiveness to all the substrates studied, followed by CMPs and HSCs. HSCs exhibits a preference for fibronectin compared to the other proteins tested. The less adhesive seems to be GMPs (a,b,c). Difference between GMPs and MEPs adhesiveness is statistically validated (p< 0.01). CD34 expression is higher on adherent cells; on the contrary, CD45RA is more expressed on non adherent cells (d,e). GMPs express more CXCR-4 and CD44 than the other subsets, MEPs exhibit high levels of α4 integrin, explaining why this population seems more adhesive to all the substrates. α4 and α5 integrins are important also for GMPs (a). Expression of CXCR-4 in all the subpopulations (b). Disclosures: No relevant conflicts of interest to declare.

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Constitutive Expression of Lef-1 in Murine Hematopoietic Progenitors Causes Myeloid and Lymphoid Leukemias Propagated by a Leukemic Stem Cell Population with Lymphoid Characteristics.

Blood ◽

10.1182/blood.v110.11.2645.2645 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2645-2645

Author(s):

Konstantin Petropoulos ◽

Christina Schessl ◽

Natalia Arseni ◽

Vijay P.S. Rawat ◽

Aniruddha Deshpande ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Population ◽

Leukemic Stem Cell ◽

Hematopoietic Stem ◽

Hematopoietic Development ◽

Blast Cells ◽

Seeding Efficiency ◽

Acute Myeloid

Abstract Canonical Wnt signaling is critically involved in normal hematopoietic development and the self-renewal process of hematopoietic stem cells. Deregulation of this pathway has been linked to a large variety of cancers including leukemia. Lef-1, a key factor of the Wnt / beta-catenin signaling pathway, plays pivotal roles in lymphoid development, but little is known about the role of Lef-1 in myeloid hematopoiesis and leukemogenesis. We now show that Lef-1 is expressed in murine hematopoietic stem cells (‘LSK’, Sca+cKit+Lin−) and both myeloid and lymphoid subpopulations as well as in different human leukemias. Using a retroviral BM transplantation model, we demonstrate that ectopic expression of wild type Lef-1 (WT) and a constitutive active mutant of Lef-1 (CA) induces a severe disturbance of normal hematopoietic development: mice transplanted with bone marrow constitutively expressing Lef-1 had a significant increase in the number of circulating myeloid cells resulting in an inverted lymphoid-myeloid ratio in the peripheral blood (ratio: 0.28 (WT), 0.10 (CA) vs. 1.07 (GFP); p<0.002). With a median latency of 12 month, transplanted mice succumbed to a lethal myeloproliferation (n=2) or acute myeloid or B-lymphoblastic leukemias (N=8). Both leukemia subtypes shared key biological characteristics such as positivity for IG DH-JH rearrangements. In addition, both subtypes were characterized by a biphenotypic cell population (B220+Mac1+Gr1+, BMG+++) as well as varying numbers of B220+Mac1−Gr1− (B+MG−) cells. In both leukemia subtypes single DJH–rearranged B+MG− cells had the highest seeding efficiency and were able to give rise to both BMG+++ and B−MG+ cells in vitro (mean seeding efficiency: B+MG−: 17.9%, vs. BMG+++: 4.7%, B−MG+: 4.0, p=0.005). Strikingly, the frequency of leukemia-initiating cells in AML was highest in the B+MG− population as determined by limit dilution transplantation assays (B+MG−: 1 in 433 vs. BMG+++/B−MG+: 1 in 8491 cells, p<0.001). Expression analysis of malignant blast cells from both lymphoid and myeloid leukemias also revealed striking commonalities with regard to the transcription profile. Blast cells from both AML and ALL diseased mice uniformly showed expression of myelo-specific genes like C/ebpα and c-fms and lymphoid specific genes E2A and Ebf-1, but lacked expression of the B-cell differentiation regulator Pax-5. These findings demonstrate that balanced expression of Lef-1 is crucial for early normal hematopoietic differentiation and that deregulation of this factor induces the development of DJH-rearranged acute myeloid and lymphoid leukemias which are propagated by a leukemic stem cell with lymphoid characteristics.

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AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates

Blood ◽

10.1182/blood-2005-09-3592 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3772-3778 ◽

Cited By ~ 142

Author(s):

André Larochelle ◽

Allen Krouse ◽

Mark Metzger ◽

Donald Orlic ◽

Robert E. Donahue ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Genetic Manipulation ◽

Retroviral Vectors ◽

Lymphoid Cells ◽

Alternative Source ◽

Hematopoietic Stem ◽

Cd34 Cells

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

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Enhanced Human Hematopoietic Stem Cell Self-Renewal Enabled by Controlling Feedback Signaling From Lineage Committed Cells

Blood ◽

10.1182/blood.v118.21.1274.1274 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1274-1274

Author(s):

Elizabeth Csaszar ◽

Daniel Kirouac ◽

Mei Yu ◽

Caryn Ito ◽

Peter W. Zandstra

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Population ◽

Ex Vivo ◽

Stem Cell Population ◽

Fed Batch ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Numbers

Abstract Abstract 1274 Clinical outcomes of hematopoietic stem cell (HSC) transplantation are correlated with infused progenitor cell dose. Limited cell numbers in a typical umbilical cord blood (UCB) unit restricts the therapeutic potential of UCB and motivates ex vivo expansion of these cells. Strategies to grow HSCs have relied on the supplement of molecules acting directly on the stem cell population; however, in all cases, sustained HSC growth is limited by the concurrent growth of more mature cells and their endogenously produced inhibitory signaling factors. Despite increasing evidence for the important role of intercellular (between cell) communication networks, the identity and impact of non-stem cell autonomous feedback signaling remains poorly understood. Simultaneous kinetic tracking of more than 30 secreted factors produced during UCB culture, including TGF-b1, MIP-1b, and MCP-1, in combination with computational simulations of cell population dynamics, enabled us to develop a global control strategy predicted to reduce inhibitory paracrine signaling and, consequently, increase HSC self-renewal. By maintaining endogenously produced ligands at specified levels using a tuneable fed-batch (automated media dilution) strategy, we achieved significant improvements in expansions of total cell numbers (∼180-fold), CD34+ cells (∼80-fold), and NOD/SCID/IL-2Rgc-null (NSG) repopulating cells (∼11-fold, detected at limiting dilution). The fed-batch strategy has been integrated into an automated bioreactor, allowing for the generation of a clinically-relevant cell product after 12 days of culture, with minimal user manipulation. As this strategy targets the HSC environment and not the stem cells directly, it has the ability to act in combination with other expansion strategies to produce synergistic results. Unexpectedly, supplementation of the soluble protein, TAT-HOXB4, to the system, yielded the expected boost in progenitor expansion only in “sub-optimal” control conditions but not in the fed-batch system. Hypothesizing that the efficacy of HOXB4 may be dependent on the skewing of supportive vs. non-supportive cell populations, and the consequent impact of paracrine ligand production, we performed kinetic tracking of 20 hematopoietic cell types during several supportive (fed-batch, HOXB4 supplemented, Notch ligand Delta1 supplemented) vs. non-supportive (control) cultures. Meta analysis of these data revealed a non-autonomous link between HOXB4, increased megakaryocyte production, and stem cell proliferation, as well as between Notch delta-1 ligand, decreased myeloid cell production, and a decrease in the growth inhibition of stem cells. These predictions have been experimentally validated using co-cultures of sorted purified HSCs and CD41+ megakaryocykes and CD14+ monocytes. Our results identify complex connections between mature cell lineages and stem cell fate decisions and we expect to report a direct link between cell-cell interactions emerging from culture manipulations and the resulting impact on HSC self-renewal. Collectively, these studies support a dominant role for non-stem cell autonomous feedback signaling in the regulation of HSC self-renewal. Overcoming cell non-autonomous inhibition of HSC self-renewal has allowed for novel strategies to enhance HSC numbers ex vivo, thereby facilitating the production of clinically relevant quantities of stem and progenitor cells and enabling more effective strategies to treat hematologic disease. Disclosures: No relevant conflicts of interest to declare.

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Life span of multipotential hematopoietic stem cells in vivo.

Journal of Experimental Medicine ◽

10.1084/jem.171.5.1407 ◽

1990 ◽

Vol 171 (5) ◽

pp. 1407-1418 ◽

Cited By ~ 137

Author(s):

G Keller ◽

R Snodgrass

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Strong Evidence ◽

Stem Cell Population ◽

Hematopoietic Stem ◽

Somatic Tissues ◽

Exogenous Factors

The findings reported in this study highlight several important features of the development of hematopoietic stem cells after transplantation into irradiated recipients. First, they demonstrate the existence of a class of primitive multipotential stem cells that can function for a significant portion of the lifetime of a mouse (15 mo). In addition, they clearly show that these primitive stem cells can be infected with recombinant retroviruses and thus would be appropriate targets for gene therapy in somatic tissues. Second, our data indicate that the progeny of some, but not all, of the primitive stem cells have fully expanded into the various hematopoietic lineages by 2 mo after reconstitution. Finally, our analysis of the secondary recipients provides strong evidence suggesting that the primitive stem cell population can actually clonally expand. Our current experiments are aimed at determining the extent to which this expansion can occur and whether or not this expansion can be influenced by exogenous factors.

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Apheresis of peripheral blood stem cells in children with very low body weight: a single centre experience

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2020-19-2-152-159 ◽

2020 ◽

Vol 19 (2) ◽

pp. 152-159

Author(s):

E. E. Kurnikova ◽

I. G. Khamin ◽

V. V. Shchukin ◽

T. V. Shamanskaya ◽

M. S. Fadeeva ◽

...

Keyword(s):

Stem Cells ◽

Body Weight ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Central Venous Access ◽

High Dose ◽

Term Survival ◽

Hematopoietic Stem ◽

Cd34 Cells

Polychemotherapy, accompanied by autologous hematopoietic stem cell transplantation, can improve the results of long-term survival of patients with cancer and some non-cancer diseases. Mobilizing and collecting hematopoietic stem cells in children with very low body weight can be a difficult task. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. 19 children with extremely low body weight was included in the current study. The median age was 8 (5–14) months, the median of body weight 7.5 (5.8–8.8) kg. Apheresis was performed in an ICU, using sedative therapy and in compliance with the conditions for the prevention of anemia, hypovolemia, hypothermia. 19 hematopoietic stem cell apheresis were performed using the Spectra Optia MNC separator program. Mobilization of CD34+ cells was performed with filgrastim; three children were additionally given plerixaphor. All 19 hematopoietic stem cell apheresis were successful: the median of collected CD34+ cells was 18.7 × 106/kg (8.6– 60.6 × 106/kg), the median apheresis duration was 204 (161–351) min. Serious side effects during apheresis were not recorded, however, in 6 children (31%) we encountered difficulties in the process of installing central venous access. The collection of hematopoietic stem cells for the future high-dose chemotherapy with autologous hematopoietic stem cells is a feasible task even for very young children with extremely low body weight. Correct preparation for manipulation, taking into account all possible risk factors and technical features, can avoid serious complications.

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Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

Blood ◽

10.1182/blood.v89.10.3596 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3596-3606 ◽

Cited By ~ 195

Author(s):

Troy D. Randall ◽

Irving L. Weissman

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Population ◽

Stem Cell Population ◽

Normal Bone Marrow ◽

Hematopoietic Stem ◽

Normal Bone

Abstract A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

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Ex Vivo Generation of CD34+ Cells From CD34− Hematopoietic Cells

Blood ◽

10.1182/blood.v94.12.4053 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4053-4059 ◽

Cited By ~ 124

Author(s):

Yoshihiko Nakamura ◽

Kiyoshi Ando ◽

Jamel Chargui ◽

Hiroshi Kawada ◽

Tadayuki Sato ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Population ◽

Cultured Cells ◽

Ex Vivo ◽

Calf Serum ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Cd34 Cells

Abstract The human Lin−CD34− cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin−CD34− cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin−CD34− cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34+ phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34+ cells in the human CD34− cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin−CD34− hematopoietic stem cells.

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Donor-Cell Derived Aplastic Anemia (AA) with a Small Population of CD55−CD59− Blood Cells after Allogeneic Stem Cell Transplantation: Evidence for the Immune System Attack Against Normal Hematopoietic Stem Cells in AA.

Blood ◽

10.1182/blood.v110.11.3682.3682 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3682-3682

Author(s):

Chiharu Sugimori ◽

Kanako Mochizuki ◽

Hirohito Yamazaki ◽

Shinji Nakao

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Immune System ◽

Stem Cell Transplantation ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Donor Cell ◽

Small Population ◽

Hematopoietic Stem

Abstract Acquired aplastic anemia (AA) is thought to be caused by the immune system attack against hematopoietic stem cells. However, there is no direct evidence that an immune system attack against normal hematopoietic stem cells leads to development of AA. The immune system attack may be directed toward abnormal stem cells given the fact that some patients with myelodysplastic syndrome respond to immunosuppressive therapy. Although the presence of a small population of CD55−CD59− blood cells represents a reliable marker for the immune pathophysiology of AA, little is known regarding when and how such paroxysmal nocturnal hemoglobinuria (PNH)-type cells appear in patients with AA. The development of AA with a small population of PNH-type cells was recently observed in an allogeneic stem cell transplant (SCT) recipient. This patient, a 59-year-old male, who had been treated with allogeneic peripheral blood stem cell transplantation (PBSCT) from an HLA-compatible sibling for treatment of very severe AA in March 2002, developed severe pancytopenia in December 2005. Late graft failure (LGF) without residual recipient cells was diagnosed based on the results of a chimerism analysis. Sensitive flow cytometry failed to reveal any increase in the proportion of CD55−CD59− PNH-type blood cells. The patient underwent a second PBSCT from the original donor without preconditioning in February 2006. Although his pancytopenia was completely resolved by day 20, his blood counts gradually decreased from day 60 without any apparent complications. Flow cytometry revealed small populations of PNH-type granulocytes in his peripheral blood (Figure 1). Both the PNH-type and normal phenotype granulocytes were of donor origin. PIG-A gene analyses showed the PNH-type granulocytes in the patient to be a clonal stem cell with an insertion of thymine at position 593 (codon 198). Similar results were obtained from the sorted PNH-type granulocytes obtained 6 months later. The patient was treated with horse antithymocyte globulin and cyclosporine. The patient required no further transfusions after 88 days of the therapy and remains well as of August, 2007. The small population of PNH-type cells was not detectable in any of 50 SCT recipients showing stable engraftment or in an AA patient suffering graft rejection after a SCT. These findings suggest that some factors expressed by the patient induced an immune system attack against autologous hematopoietic cells, leading to de novo development of donor-cell derived AA. This is the first evidence that an immune system attack against normal hematopoietic stem cells results in AA associated with a clonal expansion of a PIG-A mutant which may originally be present in the donor bone marrow. Figure Figure

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High Aldehyde Dehydrogenase Activity (ALDH) Is a General Marker for Normal Hematopoietic Stem Cells but Not Leukemic Stem Cells in Acute Myeloid Leukemia (AML). .

Blood ◽

10.1182/blood.v114.22.4035.4035 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4035-4035

Author(s):

Linda Smit ◽

Lisa A Min ◽

Monique Terwijn ◽

Angele Kelder ◽

Alexander N Snel ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Alkylating Agents ◽

Therapeutic Window ◽

Leukemic Stem Cells ◽

Aldh Activity ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Blast Cells

Abstract Abstract 4035 Poster Board III-971 Only a minority of cells, the leukemic stem cells (LSCs), within AML are responsible for tumor growth and maintenance. Many patients experience a relapse after therapy which is thought to originate from the outgrowth of therapy resistant LSC. Therefore, eradication of the LSCs is likely necessary to cure AML. Both the normal hematopoietic stem cells (HSCs) and LSCs co-exist in the bone marrow (BM) of leukemia patients and therefore success of an anti-stem-cell strategy relies on specific induction of LSC death while sparing the normal HSC. In AML, apart from the CD34+CD38- and the side population (SP) compartment, the high ALDH activity compartment contains the LSCs. The SP and ALDH defined compartments may include both CD34+ and CD34- HSCs and LSCs. ALDH is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes and high ALDH activity results in resistance to alkylating agents such as the active derivatives of cyclophosphamide. Recent data has shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blast cells. In view of the applicability of LSC specific therapies the detoxification by ALDH might be of importance. Therefore, a difference in ALDH activity between the normal HSC and the malignant LSC might be used to preferentially kill the LSC and spare the HSC. We have previously shown that CD34+CD38- and SP LSCs can be identified and discriminated from HSCs using stem cell-associated cell surface markers, including C-type lectin-like molecules (CLL-1), lineage markers, such as CD7, CD19, and CD56 and recently cell size characteristics as measured by flow cytometry (Terwijn, Blood 111: 487,2008). Here we have analyzed ALDH activity in 23 AML cases. In 7 AML cases, a high SSCloALDHbr cell population was identified (median: 10,9%, range 5,24-15,29%). In 16 cases there were rare (<5%) SSCloALDHbr cells. We have analyzed ALDH activity in aberrant marker defined HSCs and LSCs, both present within the same BM samples in 18 AML patients (summarized in Figure 1). In 9 BM AML samples, defined as CD34-, the CD34+ compartment contained only normal CD34+CD38- HSCs. The ALDH activity in the CD34- cells, which includes by definition in this AML subgroup the LSC, is a factor 4,4 (range 1,7-18,9) lower than in the HSC (Figure 1, panel 1). The ALDHbrSSClo cells present in these CD34- AML cases contained both normal CD34+ and CD34- cells. The activity of the normal HSC within this AML BM is similar to that of the normal HSC in NBM of healthy donors (Figure 1, panel 3). In addition, 9 BM AML patients, defined as CD34+ AML and with both marker negative, normal HSCs and marker positive LSCs present, were analyzed for ALDH activity. We show that the marker positive CD34+CD38- LSCs have 7,7 fold (range 1,73-29,2 fold) lower ALDH activity than the marker negative CD34+CD38- HSCs (Figure 1, panel 2). Altogether, we show that, although malignant AML blast cells have varying ALDH activity, a common feature of all AML cases is that the normal HSCs that co-exist with leukemic (stem) cells in the BM of AML patients have a higher ALDH activity as compared to their malignant counterparts (summarized in figure 1). In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM (irrespective of AML phenotype, CD34+ or CD34-) at diagnosis. Consequently, AML patients with high ALDH activity in the normal HSC might benefit from treatment with alkylating agents such as cyclophosphamide, whereby the difference between the ALDH activity in LSC and HSC defines the therapeutic window. At present, drugs, known to be dependent on low ALDH for proper activity, are tested for their LSC specific killing while sparing the normal HSC. Additionally, transcriptional profiles are obtained from purified ALDH+ HSC and ALDH- LSC. This will enable us to use this general discriminating property to identify molecules that differ between the LSC and HSC and can function as LSC specific therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

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