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

Blood ◽  
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.

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

Blood ◽  
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.

Superior Impact of p18INK4c over p27kip1 on Long-Term Repopulating Ability of Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 796-796
Author(s):  
Hui Yu ◽  
Hongmei Shen ◽  
Xianmin Song ◽  
Paulina Huang ◽  
Tao Cheng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Impact

Abstract The G1-phase is a critical window during the cell cycle in which stem cell self-renewal may be balanced with differentiation and apoptosis. Increasing evidence suggests that the cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1/Waf1, p27kip1, p16INK4A, and p18INK4C (p21, p27, p16 and p18 hereafter) are involved in stem cell self-renewal, as largely demonstrated in murine hematopoietic stem cells (HSCs). For example, we have recently demonstrated a significant increase of HSC self-renewal in the absence of p18 (Yuan et al, Nature Cell Biology 2004). But the actual roles of these CKIs in HSCs appear to be distinct as p21 and p18 have opposite effects (Yu H et al, ASH 2004) whereas p16 has a limited effect (Stepanova et al, Blood 2005) on HSC exhaustion after serial bone marrow transfer. Like p18, however, p27 was recently reported to also inhibit HSC self-renewal due to the fact that the competitive repopulating units (CRUs) were increased in p27−/− mouse bone marrow (Walkley et al, Nature Cell Biology 2005) in contrast to the results in a previous report (Cheng T et al, Nature Medicine 2000). To further gauge the impact of p18 versus p27 on the long-term repopulating ability (LTRA) of HSCs, we have generated different congenic strains (CD45.1 and CD45.2) of p18−/− or p27−/− mice in the C57BL/6 background, allowing us to compare them with the competitive repopulation model in the same genetic background. The direct comparison of LTRA between p18−/− and p27−/− HSCs was assessed with the competitive bone marrow transplantation assay in which equal numbers of p18−/− (CD45.2) and p27−/− cells (CD45.1) were co-transplanted. Interestingly, the p18−/− genotype gradually dominated the p27−/− genotype in multiple hematopoietic lineages and p18−/− HSCs showed 4-5 times more LTRA than p27−/− HSCs 12 months after cBMT. Further self-renewal potential of HSCs was examined with secondary transplantation in which primarily transplanted p18−/− or p27−/− cells were equally mixed with wild-type unmanipulated cells. Notably, while the p18−/− cells continued to outcompete the wild-type cells as we previously observed, the p27−/− cells did not behave so in the secondary recipients. Based on the flow cytometric measurement and bone marrow cellularity, we estimated that transplanted p18−/− HSCs (defined with the CD34−LKS immunophenotype) had undergone a 230-fold expansion, while transplanted p27−/− and wild-type HSCs had only achieved a 6.6- and 2.4-fold expansion in the secondary recipients respectively. We further calculated the yield of bone marrow nucleated cells (BMNCs) per HSC. There were approximately 44 x 103, 20.6 x 103, and 15 x 103 BMNCs generated per CD34−LKS cell in p18−/−, p27−/− and wild-type transplanted recipients respectively. Therefore, the dramatic expansion of p18−/− HSCs in the hosts was not accompanied by decreased function per stem cell. Our current study demonstrates that hematopoietic engraftment in the absence of p18 is more advantageous than that in the absence of p27, perhaps due to a more specific role of p18 on self-renewal of the long-term repopulating HSCs.

scholarly journals Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo

2019 ◽  
Vol 116 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Du Jiang ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Different Levels

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

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

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3772-3778 ◽  
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.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

CD166 (ALCAM): A Functional Marker of Primitive Murine and Human Hematopoietic Stem Cells and Cellular Elements of Their Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 640-640
Author(s):  
Brahmananda Reddy Chitteti ◽  
MIchihiro Kobayashi ◽  
Yinghua Cheng ◽  
Peirong Hu ◽  
Bradley Poteat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Immunoglobulin Superfamily ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Normal Ranges ◽  
The Impact ◽  
Hematopoietic Niche

Abstract Abstract 640 Phenotypic definition of murine and human hematopoietic stem cells (HSC) relies on a large number of markers. Few surface antigens with functional importance have been identified as key common markers on adult murine and human HSC. Intimate interactions between HSC and elements of the hematopoietic niche (HN) depend on ligand-counter ligand molecules co-expressed independently on niche components or putative HSC. To date, not a single surface marker has been identified as a common marker expressed on murine and human HSC and on cells of the HN. We previously demonstrated that CD166 is expressed on osteoblasts (OB) that mediate a hematopoiesis enhancing activity (HEA). Given that CD166 is a member of the immunoglobulin superfamily that can mediate homophilic cell-cell interactions, we investigated the role of CD166 in identifying HSC and the impact of CD166 on hematopoiesis, stem cell engraftment, and the HN. Interestingly, CD166+, but not CD166- fractions of murine and human repopulating HSC identified by a rigorous hierarchical classification for each species mediated robust long-term engraftment. In the murine system, 25 sorted Lineage- Sca1+ ckit+ (LSK) CD48-CD150+CD166+ cells mediated 69.5 ± 7.3% chimerism 4 months post-transplantation (PT) while donor-derived chimerism supported by 25 sorted LSKCD48-CD150+CD166- cells was only 13.6 ± 11.6% (p<0.01) suggesting that CD166 identifies long-term repopulating cells beyond what is possible with SLAM markers. In the human system, 1000 cord blood-derived Lin-CD34+CD38-CD49f-CD166+ cells and Lin-CD34+CD38-CD49f+CD166+ cells engrafted at 44.5 ± 9.7% and 38.4 ± 8.9%, respectively 16 weeks PT in conditioned NSG mice. More importantly, chimerism derived from Lin-CD34+CD38-CD49f+CD166- cells was 1.6 ± 0.1% (p<0.01 vs both fractions) demonstrating that the CD166+ but not the CD166- fraction of CD34+Lin-CD38- cells (regardless of the status of CD49f expression) contains long-term engrafting human HSC. In CD166 knockout (KO) mice, numbers of LSKCD48-CD150+ cells in the bone marrow and Lin-CD48- cells in the peripheral blood were significantly reduced relative to wild-type (WT) controls although other hematopoietic parameters in KO mice were within normal ranges. Phenotypically defined HSC from CD166−/− mice failed to engraft in lethally irradiated WT recipients. Levels of engraftment 4mo PT of 1,000 LSK cells from WT donors into WT recipients was 71.8% ± 8.3% while that obtained from a similar number of KO cells was 5.8% ± 2.8% (p<0.01). To permit direct comparison of KO and WT mice as recipients, both genotypes were transplanted with purified LSK cells from GFP C57BL/6 mice. While short-term repopulating GFP cells engrafted efficiently in KO mice 1mo PT (66.0% ± 6.5%), reconstitution declined substantially 2mo PT and was 10.3% ± 2.7% at 3mo PT (compared to 52.6% ± 10.4% in WT hosts, p<0.01) and less than 5% at 4mo PT, demonstrating that the CD166−/− hematopoietic niche can not support long-term repopulating cells. We used our previously described co-culture system to assess the impact of homophilic CD166 interactions on the HEA of OB. The highest HEA was reached when both OB and LSK cells expressed CD166. However, when either or both cell types lacked CD166 expression, the degree of HEA was significantly lower demonstrating that homophilic CD166 interactions are critical to maintaining HSC function. Since Stat3 has 3 binding sites on the CD166 promoter, we examined the relationship between expression of Stat3 and CD166. HSC from Stat3−/− mice which do not engraft efficiently in WT recipients expressed very low levels of CD166. In addition, pharmacologic inhibition of Stat3 expression led to a simultaneous inhibition of CD166 expression. Reconstitution kinetics data and survival of KO mice under hematopoietic stress conditions suggested that CD166−/− HSC have an intrinsic self-renewal capacity precluding them from both rapid proliferation and expansion and maintenance of the stem cell pool in the HN. Our data illustrate for the first time, that CD166 is a universal marker of both murine and human HSC and OB within the HN and suggest that CD166 may modulate HSC-niche interactions and impact stem cell fate. The conserved homology between murine and human CD166 provides an excellent bridge between human and murine studies for efficient translational investigations and interventions for enhancing stem cell engraftment and clinical utility. Disclosures: Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy.

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

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.

scholarly journals Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

2019 ◽  
Vol 3 (15) ◽  
pp. 2355-2367 ◽  
Author(s):  
Yueh-Chwen Hsu ◽  
Tsung-Chih Chen ◽  
Chien-Chin Lin ◽  
Chang-Tsu Yuan ◽  
Chia-Lang Hsu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Knockout Mice ◽  
Neurodevelopmental Disorder ◽  
Loss Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Knockout Mouse Model ◽  
The Impact

Abstract Plant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

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

Blood ◽  
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.

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 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document