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

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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

Blood ◽

10.1182/blood.v108.11.1897.1897 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1897-1897

Author(s):

Ramon V. Tiu ◽

Jennifer J. Powers ◽

Abdo Haddad ◽

Ying Jiang ◽

Jaroslaw P. Maciejewski

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Population ◽

Stem Cell Population ◽

Leukemic Stem Cell ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Slam Family

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.

Download Full-text

Novel Lentiviral Vectors Displaying ‘Early-Acting-Cytokines’ Preferentially Promote the Survival and Transduction of NOD/SCID Repopulating Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.2107.2107 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2107-2107

Author(s):

E.L.S. Verhoeyen ◽

Maciej Wiznerowicz ◽

Delphine Olivier ◽

Brigitte Izac ◽

Didier Trono ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Stem Cell ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Lentiviral Vectors ◽

Hematopoietic Stem ◽

Efficient Gene ◽

Cd34 Cells

Abstract A major limitation of current generation lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent target cells which hampers their application for hematopoietic stem cell gene therapy. Human CD34+ cells that reside into G0 phase of the cell cycle and thus are quiescent, are indeed higly enriched in hematopoietic stem cells. Here, we designed novel lentiviral vectors that overcome this type of restriction by displaying early-acting-cytokines on their surface. Presentation of a single cytokine, thrombopoietin (TPO), or co-presentation of TPO and stem cell factor (SCF) on the lentiviral vector surface improved gene transfer into quiescent CD34+ cord blood cells by 45-fold and 77-fold, respectively, as compared to conventional lentiviral vectors. Moreover, these new LVs preferentially transduced and promoted the survival of immature resting cells rather than cycling CD34+ cells. Most importantly, the new early-cytokine-displaying lentiviral vectors allowed highly efficient gene transfer in CD34+ immature cells with long-term in vivo NOD/SCID mice repopulating capacity, a hallmark of bona fide HSCs. In conclusion, the novel ‘early-acting cytokines’ displaying LVs described here provide simplified, reproducible gene transfer protocols that ensure efficient gene transfer in hematopoietic stem cells. As such, these novel reagents bring us one step closer to selective in vivo gene therapy.

Download Full-text

Aldehyde Dehydrogenase Expression in Primitive Human Hematopoietic Progenitor Cells with Side Population Characteristic and in Samples from Patients with Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v106.11.4570.4570 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4570-4570

Author(s):

Ute Meissauer ◽

Joerg Hoffmann ◽

Christian Scharenberg ◽

Andreas Neubauer ◽

Cornelia Brendel

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Staining Pattern ◽

Side Population ◽

Aldh Activity ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Blast Cells

Abstract High activity of the detoxifying enzyme aldehyde dehydrogenase (ALDH) has been attributed to primitive hematopoietic stem cells in the murine and human system. ALDH activity can be measured by a functional assay employing BODIPY-aminoacetaldehyde (BAAA) as a fluorescent intracellular substrate. High ALDH activity within hematopoietic stem cells is associated with CD34 and AC133 surface expression. Given the co-expression of these markers in acute myeloid leukemia (AML), we sought to investigate whether ALDH-activity can be detected in early blood stem cell disorders and in primitive hematopoietic progenitor cells with the side population phenotype. Employing a commercial Aldefluor™ -kit thirteen peripheral blood and bone marrow samples from patients with AML were stained for ALDH activity. Four samples exhibited a clearly distinctive positive staining pattern when compared to the inhibitor control, i.e. 16–91% of AML blast cells were considered as ALDH positive. The staining pattern was similar to marrow samples from healthy donors or peripheral blood sample controls from G-CSF-mobilised healthy stem cell donors. In addition we investigated ALDH activity in primitive bone marrow side population (SP) cells from healthy individuals. Although the ALDH substrate BAAA is actively extruded out of cells and especially SP stem cells by an ABC-transporter-activity, we established a co-staining method for Hoechst 33342 and BAAA by inclusion of specific ABC-transporter inhibitors. Both SP cells and ALDH-positive cells appeared as a clear distinctive population with some overlap between both populations. Quantitative RT-PCR of different sorted stem cell samples confirmed that SP cells had higher ABCG2-expression than ALDH positive cells and ABCG2-expression was higher in ALDH positive cells compared to the total cell fraction or granulocytes. We conclude that ALDH is expressed in some but not all AML blast cells. This is in accordance with the cytogenetic heterogeneity of this disease and may have therapeutic implications because of the detoxifying activity of the ALDH enzyme. In addition ALDH activity can also be found on a minor population of very primitive marrow progenitor cells. Whether high ALDH activity is a feature of the leukemic stem cell requires further investigation.

Download Full-text

Generation Of FANCA-/- Human CD34+ Hematopoietic Stem Cells By shRNA Knockdown

Blood ◽

10.1182/blood.v122.21.2903.2903 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2903-2903

Author(s):

Zejin Sun ◽

Rikki Enzor ◽

Paula Rio ◽

D. Wade Clapp ◽

Helmut Hanenberg

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Lentiviral Vector ◽

Human Fibroblasts ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Control Shrna ◽

Cd34 Cells

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow (BM) failure, genomic instability, and a predisposition to malignancies. Natural gene therapy due to molecular self-correction of hematopoietic stem cells (HSCs) has been reported in a minority of FA patients, suggesting that due to the in vivo selection advantage of the corrected cells, FA is an excellent model disease for stem cell gene therapy. However, the scarcity of autologous HSCs from FA patients for research purposes is one of the major road blocks to preclinical studies with human cells. Here, we developed a lentiviral vector with EGFP as marker gene that co-expresses two distinct shRNA sequences against FANCA under two different human promoters (H1 and U6). In vitro analysis in primary human fibroblasts showed that stable integration of this construct was highly efficient to induce the typical FA cellular phenotypes as assessed by (1) FANCD2 ubiquitination deficiency and (2) a characteristic G2/M arrest upon DNA damage induced by DNA crosslinking reagent Mitomycin C (MMC). We then transduced human cord blood (CB) CD34+ cells with this lentiviral vector and demonstrated a reduced survival of clonogenic cells in progenitor assays at 20nM MMC: 70% (scrambled control shRNA) vs. 23% (FANCA shRNA). This vector pseudotyped with a foamyviral envelope was then used to transduce CD34+ CB cells on fibronectin CH296. The next day, genetically modified cells were transplanted into NOD.Cg---Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. When analyzing the percentage of EGFP+ cells in the human graft (hCD45+ cells), we noticed a progressive decline of EGFP+ cells from 29% on day 5 to 5% at 4 months after transplantation in the peripheral blood of the recipient mice, mimicking the progressive BM failure in FA patients. In contrast, engraftment over time was stable in CD34+ cells transduced with scrambled control shRNA vector (33% on day 5 vs. 34% at 4 months). The human progenitors isolated from the BM of NSG recipient mice at sacrifice 4 months after initial transduction and transplantation are still hypersensitive to MMC, with a much lower survival rate of 34% at 20nM MMC in the FANCA shRNA group as compared to 78% in the scrambled control shRNA group, thus confirming the knockdown by the lentiviral shRNA construct is stable. In summary, the novel double shRNA lentiviral vector is capable of inducing all major hallmarks of FA cells in normal human CB CD34+ cells, thus providing unlimited FA-like cellular materials including NSG mice-repopulating HSCs for preclinical gene therapy and basic stem cell biology research in FA. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis

Journal of Experimental Medicine ◽

10.1084/jem.20042541 ◽

2005 ◽

Vol 202 (1) ◽

pp. 169-179 ◽

Cited By ~ 116

Author(s):

Yuko Kato ◽

Atsushi Iwama ◽

Yuko Tadokoro ◽

Kazuya Shimoda ◽

Mayu Minoguchi ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Leukemic Stem Cells ◽

Myeloproliferative Disease ◽

Self Renewal ◽

Hematopoietic Stem

Although the concept of a leukemic stem cell system has recently been well accepted, its nature and the underlying molecular mechanisms remain obscure. Constitutive activation of signal transducers and activators of transcription 3 (STAT3) and STAT5 is frequently detected in various hematopoietic tumors. To evaluate their role in normal and leukemic stem cells, we took advantage of constitutively active STAT mutants to activate STAT signaling selectively in hematopoietic stem cells (HSCs). Activation of STAT5 in CD34–c-Kit+Sca-1+ lineage marker– (CD34–KSL) HSCs led to a drastic expansion of multipotential progenitors and promoted HSC self-renewal ex vivo. In sharp contrast, STAT3 was demonstrated to be dispensable for the HSC maintenance in vivo, and its activation facilitated lineage commitment of HSCs in vitro. In a mouse model of myeloproliferative disease (MPD), sustained STAT5 activation in CD34–KSL HSCs but not in CD34+KSL multipotential progenitors induced fatal MPD, indicating that the capacity of STAT5 to promote self-renewal of hematopoietic stem cells is crucial to MPD development. Our findings collectively establish a specific role for STAT5 in self-renewal of normal as well as leukemic stem cells.

Download Full-text

Targeting miRNA-551b, a "Stemness"-like microRNA, to Eradicate AML (Stem) Cells

Blood ◽

10.1182/blood-2018-99-116024 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1494-1494

Author(s):

Tània Martiáñez ◽

Noortje Van Gils ◽

David Christian De Leeuw ◽

Eline Vermue ◽

Arjo Rutten ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Inhibition Of Proliferation ◽

Novel Target

Abstract Despite high complete remission (CR) rates achieved after chemotherapy, only 30-40% of patients with Acute Myeloid Leukemia (AML) survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy-resistant leukemia cells with stem cell properties, named "leukemic stem cells" (LSCs). LSCs use a variety of mechanisms to resist chemotherapy and targeting them is one of the major challenges in AML treatment. Since miRNAs can target multiple genes/pathways simultaneously, their modulation (downregulation or upregulation) may have great potential for the successful elimination of therapy-resistant leukemic (stem) cells (Martiañez Canales et al. Cancers 2017). Here, we show that miRNA-551b, previously identified by us as a stem cell-like miRNA, can be a potential novel target to specifically eradicate AML stem-like cells. Aiming at identification of miRNA-based therapy to specifically eradicate LSCs, while sparing normal Hematopoietic Stem Cells (HSCs), we determined expression of miRNAs in normal HSCs, Leukemic Stem Cells (LSCs) and leukemic progenitors (LP) all derived from the same AML patient's bone marrow. Using this approach, we identified miRNA-551b as being highly expressed in normal HSCs residing both in healthy and AML bone marrows. In AML, high expression of miR551b demonstrated to be associated with an adverse prognosis. Moreover, miRNA-551b was highly expressed in immature AML cases and its expression in a cohort of patients coincided with the expression of stem cell genes (De Leeuw et al. Leukemia 2016). To further elucidate the link between miRNA-551b and AML "stemness" and to test whether downregulation of miRNA-551b affects the survival of AML (stem/progenitor) cells, proliferation and the balance between differentiation and "stemness", we reduced miRNA-551b expression, either by lentiviral transduction of antagomirs or by adding locked nucleotide acid (LNA)-oligonucleotides to AML cell lines and primary AML cells. Downregulation of miRNA-551b in the stem cell-like AML cell line KG1a led to inhibition of cell growth in vitro, which was due to inhibition of proliferation rather than induction of apoptosis. KG1a tumor growth in an in vivo mouse model was also reduced when miRNA-551b was downregulated. In primary AML, miRNA-551b knockdown resulted in a significant decrease in the survival of leukemic progenitors and LSCs, while hematopoietic stem cells (HSCs) and normal progenitors from healthy bone marrows were not affected. These results suggest that a therapeutic approach inhibiting miRNA-551b expression might specifically eradicate leukemic progenitors and LSCs from primary AML, while sparing HSCs. We are currently studying miRNA-551b targets which can be responsible for this specific LSCs elimination. In conclusion, our results suggest that inhibition of miRNA-551b could be a promising approach to eliminate stem cell-like AML cells, thereby decreasing relapse rates and improving AML treatment outcome. Disclosures Ossenkoppele: Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.

Download Full-text

Autologous Stem Cell Transfusions on Multiple Days in Patients with Multiple Myeloma - Does It Matter?

Blood ◽

10.1182/blood-2019-127940 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3252-3252

Author(s):

Thomas Pabst ◽

Sebastian Moser ◽

Ulrike Bacher ◽

Barbara Jeker ◽

Behrouz Mansouri Taleghani ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

High Dose ◽

Hematopoietic Stem ◽

Increased Risk ◽

Cd34 Cells ◽

The Impact

Introduction: Autologous stem cell transplantation (ASCT) following high-dose chemotherapy (HDCT) is a cornerstone in the standard first-line treatment in myeloma (MM) patients. Freezing of the hematopoietic stem cells (HSC) to bypass the time between stem cell collection and completion of HDCT is crucial for this process. Due to the vulnerability of HSC, adding of anti-freezing agents such 5-10 vol% dimethyl-sulfoxide (DMSO) to hematopoietic stem cells is mandatory. DMSO exerts toxic effects after administration, and toxicity of DMSO is dose-related. However, guidelines for this procedure are missing, and transplant centers have implemented varying limitations of maximum total DMSO administration, ranging from 20-70 g per day. At our center, the maximum transplant volume is 300 mL per day with DMSO at 5 vol%. For patients with transfusion volumes above these limits, the transplant procedure is split over several days. Methods: In this single center study, we retrospectively analyzed the impact of multiple day transplantation procedures on survival rates and hematological recovery in 271 patients with MM patients undergoing first melphalan-based ASCT. Results: 244 (90%) received ASCT within a single day, and this group was termed Tx1. The Tx2-3 group comprised 23 patients receiving stem cells on 2 days, and four patients on 3 days. Both groups (Tx1 and Tx2-3) did not differ in clinical characteristics or number/types of induction therapy lines. The remission status pre-transplant was comparable. Plerixafor was given more frequently in Tx2-3 than Tx1 (p=0.0715). At the day of SC collection, peripheral CD34+ counts were lower in Tx2-3. The final administered autograft volume was higher in Tx2-3 patients. The amount of transplanted CD34+ cells/kg b.w. was lower in the Tx2-3 group, mirroring poorer mobilization of CD34+ cells (p<0.0001). The median recovery for neutrophils was 13 days for Tx2-3 and 12 days for Tx1 (p=0.0048), and for platelets 18 versus 14 days (p=0.0004). Tx2-3 patients had longer median hospitalization duration (23 versus 19 days; p=0.0006). The median follow-up was 56 months. Relapse-free survival (RFS) was 39 months, and 169 relapses (62%) occurred so far. Median OS was 91 months, and 82 patients (30%) have died during follow-up. Tx2-3 patients had shorter median RFS (21 versus 40 months for Tx1; p=0.0245), and shorter median OS with 55 versus 93 months (p=0.0134) (Figure 1). Conclusions: Our data suggest that multiple day transplantation is associated with poor CD34+ mobilization and is observed in roughly 10% of myeloma patients. Patients with multiple day transplant procedures had later neutrophil and platelet engraftment, longer hospitalization duration, more febrile episodes, and inferior OS and RFS. This suggests to consider myeloma patients with the need for multiple day transplantation as a patient group at increased risk that needs enhanced surveillance strategies. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Stringent Small Molecule Dose Requirements for the Optimal Expansion of Hematopoietic Stem Cells Revealed By Predictive Analytics and Xenotransplants

Blood ◽

10.1182/blood-2019-128692 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1185-1185

Author(s):

Javed K Manesia ◽

Sakhar Almoflehi ◽

Roya Pasha ◽

Suria Jahan ◽

John Blake ◽

...

Keyword(s):

Stem Cells ◽

Ascorbic Acid ◽

Stem Cell ◽

Cell Growth ◽

Hematopoietic Stem Cells ◽

Small Molecules ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

The Impact

Introduction: Loss of self-renewal of hematopoietic stem cells (HSC) is a major roadblock to cell engineering therapies. Small molecules have been identified that promote HSC expansion. We recently identified UM171, StemReginin1 (SR1) and valproic acid (VPA) as strongest agonist for expansion of cord blood (CB) CD34+CD45RA- and CD34+CD45RA-EpcrHigh (EpcrHg) HSC-enriched cells of 12 molecules tested. In addition, we identified a novel putative stem cell agonist in L-Ascorbic acid 2-phosphate (AA2P), a derivative of vitamin C. Using response surface methodology and machine learning, we identified a series of Stem Cell Agonist Cocktails (SCAC) composed of these 4 agonists at varying concentrations. The objectives of this study were to characterize the in vitro properties of AA2P and SCACs on CB HSC and, test the capacity of AA2P- and SCAC-expanded CB CD34+ cells to support hematopoietic recovery and long-term (LT) engraftment after transplantation. Methods: Predictive models for HSC expansion (CD34+CD45RA- and EpcrHg) promoted by SR1, UM171, VPA, and AA2P were built by design of experiments. The data was then used to train a neural network. These were used as predictive tools to derive a series of SCAC composed of different concentrations of the 4 agonists (Table 1). CB expanded HSPC were characterized after 14 days of culture. Migration of HSPCs toward SDF-1 was tested in a transwell assay. Serial and limit dilution transplant assays in NSG mice were done to characterize the capacity of SCAC to support expansions of short-term (ST) and LT HSC. Results: First, we investigated the capacity of AA2P to act as an HSC agonist. AA2P was unable on its own to expand EpcrHg cells but promoted cell growth and the expansion of CD34+CD45RA- HSPC (2-fold, p<0.05), a property shared by L-Ascorbic acid. Moreover, AA2P-expanded HSPCs enhanced ST platelet engraftment when compared to serum-free medium (SFM) control (p=0.053, n=3). Next, we tested the activity of SCACs presented in Table 1. Varying the concentrations of the small molecules profoundly impacted cell growth and the type of HSPC expanded (Table 1). For instance, SM-2 with high UM171 provided high expansion of EpcrHg, but low level of overall cell growth. SM-A and SM-6 maximized expansion of CD34+CD45RA- cells but had lower expansion of EpcrHg due partly to lower UM171. X2A was unique as it produced balanced expansion of EpcrHg and CD34+CD45RA- cells. Lowering AA2P concentration in X2A significantly reduced the expansion of both HSC-enriched fractions (X2B, Table 1). Moreover, most SCACs enhanced expansion of HSC-enriched cells (CD34+CD45RA-CD38-CD90+CD133+, p<0.05 vs SFM) and that of lymphoid-primed multi-potential progenitors and multipotent progenitors vs. SFM cultures (p<0.05), but not of downstream progenitors. Since homing to the bone marrow (BM) is a key step towards engraftment, we investigated whether SCACs influenced the expression of homing receptors and the migration activity of HSPCs. SCAC expanded HSPCs were characterized with elevated fucosylation of PSGL-1 known to favor homing and engraftment (e.g. 82 ± 2% vs. 42 ± 8% for X2A vs. SFM CD34+ cells, p<0.01, n=4). Also, most SCAC-expanded CD34+ cells showed improved migration towards SDF-1 (e.g. 22 ± 6% vs. 13 ± 9% for X2A vs. SFM CD34+ cells, p<0.05, n=4). The capacity of SCAC-expanded HSPCs to support engraftment is still ongoing. Current results showed that X2A-expanded HSPCs provided the strongest ST recovery of platelets and leucocytes of all SCACs-HSPC, superior also to that seen with UM171-expanded HSPCs and non-cultured HSPCs (p<0.05, n=2-3). Further, LT human BM reconstitution was notably better for X2A- and SM-6-expanded HSPCs than other SCAC-expanded cells (p<0.05 vs SM2, n=2). Moreover, reducing AA2P in X2A resulted in a loss in ST and LT engraftment activity (p<0.05, n=2). Secondary transplants and limit dilution assays are ongoing to provide further insights into the impact of SCACs on the production and self-renewal activity of HSCs. Conclusion: Our study reveals that AA2P promotes cell growth and can synergize with strong stem cell agonists to promote the expansion of ST and LT engrafting HSPCs. The engraftment properties of SCAC-expanded HSPCs was highly dependent on the concentrations of the small molecules due in part to negative interactions amongst some of the agonists. Gene expression studies are ongoing to define the transcriptional landscape of HSPC produced with these SCACs. Disclosures No relevant conflicts of interest to declare.

Download Full-text