Flow cytometric enumeration of CD34+ hematopoietic stem cells: A comparison between single- versus dual-platform methodology using the International Society of Hematotherapy and Graft Engineering protocol

Abstract Abstract 848 Introduction: Acute myeloid leukemia (AML) arises from a malignant stem cell population that is resistant to cytotoxic therapy and represents a critical reservoir of conferring disease recurrence. A major focus of investigation is the identification of unique markers on leukemia stem cells (LSCs) that differentiate them from normal hematopoietic stem cells and thereby serve as potential therapeutic targets. MUC1 is a high molecular weight transmembrane glycoprotein that is aberrantly expressed in many epithelial tumors and confers cell growth and survival. We have developed an inhibitor of the MUC1-C receptor subunit that blocks oligomer formation and nuclear localization. In the present study, we have examined expression of MUC1 on LSCs as compared to normal hematopoietic stem cells and studied the effect of MUC1-C inhibition on the functional properties of LSCs. Methods and Results: Using multichannel flow cytometric analysis, we isolated the LSC compartment as defined by CD34+/CD38-/lineage- cells from bone marrow specimens obtained from patients with active AML. The majority of LSCs strongly expressed MUC1 with a mean percentage of 77% (n=6). These findings were confirmed by immunocytochemical staining of LSCs isolated by flow cytometric sorting. MUC1 expression was not detectable on the CD34- fraction of AML cells, but was present on the granulocyte-macrophage progenitor (GMP) fraction (CD34+/CD38+ cells) (mean=83%; n=6). In contrast, MUC1 expression was not observed on CD34+ progenitors isolated from normal donors (18%, n=6). In concert with these findings, RT-PCR analysis for MUC1 RNA demonstrated expression in CD34+ cells isolated from AML patients, but not normal volunteers. Notably, we also found that MUC1 expression selectively identifies malignant hematopoietic progenitors in a patient with chimerism between normal and leukemia derived stem cells. The presence of MUC1+CD34+ cells was detected in a patient with AML who achieved a morphologic complete remission following sex mismatched allogeneic transplantation. Using Bioview technology, we found that MUC1 is expressed only in the recipient (XX) CD34+ cells, representing residual malignant cells, whereas the donor (XY) derived CD34+ cells, representing the majority of the progenitors, lacked MUC1 expression. We subsequently examined the effects of MUC1-C inhibition on the capacity of leukemic progenitors to proliferate and support colony formation. MUC1-C inhibition with the GO-203 cell-penetrating peptide resulted in downregulation of the β-catenin pathway, an important modulator of cell division and survival, which is known to support the LSC phenotype. No significant change was detected with a control peptide, or with MUC1-C inhibition of progenitors isolated from a normal control. Furthermore, MUC1-C inhibition resulted in apoptosis, as demonstrated by flow cytometric staining for AnnexinV in AML CD34+ cells, but not in CD34+ progenitors isolated from normal volunteers (mean Annexin positive cells 53% and 5%, respectively, n=4). Consistent with these findings, the MUC1-C inhibitor, but not the control, peptide resulted in cell death of CD34+ cells isolated from AML patients, but not normal controls. Most significantly, exposure of CD34+ AML cells to the MUC1-C inhibitor resulted in loss of their capacity for colony formation in vitro with mean colonies of 4 and 40 for those cells exposed to the MUC1 inhibitor and a control peptide (n=2). In contrast, colony formation by normal hematopoietic stem cells was unaffected. Conclusions: MUC1 is selectively expressed by leukemic progenitors and may be used to differentiate malignant from normal hematopoietic stem cell populations. MUC1-C receptor subunit inhibition results in (i) downregulation of b-catenin signaling, (ii) induction of apoptosis and cell death, and (iii) disruption of the capacity to induce leukemia colony formation. Disclosures: Stone: genzyme: Consultancy; celgene: Consultancy; novartis: Research Funding. Kufe:Genus Oncology: Consultancy, Equity Ownership.

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The CXCR4 Antagonist AMD 3100 Induces Rapid Mobilization of Facilitating Cells and Hematopoietic Stem Cells in Mice

Blood ◽

10.1182/blood.v118.21.4694.4694 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4694-4694

Author(s):

Hong Xu ◽

Ziqiang Zhu ◽

Yiming Huang ◽

Suzanne T. Ildstad

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Fold Increase ◽

Dose Titration ◽

Great Promise ◽

Hematopoietic Stem ◽

Flow Cytometric

Abstract Abstract 4694 Bone marrow transplantation (BMT) offers great promise for treating red blood cell disorders, inherited disorders of metabolism, autoimmune diseases, and inducing donor-specific tolerance to organ transplants. However, the widespread application of this approach is dependent upon the development of less toxic strategies for BMT and avoidance of graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FC) facilitate engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex barriers without causing GVHD. We previously reported that Flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) synergistically mobilize FC and HSC into the peripheral blood (PB). Recently, AMD 3100 has been found to be a rapid mobilizing agent whose effect occurs within hours after injection. It is a macrocyclic compound and potential fusion inhibitor that antagonizes CXCR4 alpha-chemokine receptor for its effect on HSC mobilization. CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), are important in HSC homing and maintenance in the bone marrow microenvironment. Here, we investigated the effects of AMD 3100 on the mobilization of FC and HSC into PB in combination with FL and G-CSF. A dose titration of AMD 3100 was first performed. B6 mice were injected subcutaneously with AMD 3100 with the doses ranging from 1.25 mg/kg to 10 mg/kg. PB was obtained 0.5, 1, 3, and 6 hours post-injection. After individual count of peripheral blood mononuclear cells (PBMC), cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−). The numbers of PBMC significantly increased even 0.5 hour after AMD 3100 treatment and peaked at 1 h. The maximal mobilization of PBMC was noted at 1 h with 5.0 mg/kg AMD 3100. Treatment with 5.0 mg/kg AMD 3100 caused a 3.1-fold increase of WBC at 1h compared with saline treated controls. An increase of FC was detectable with all doses of AMD 3100. The numbers of FC peaked between 1 and 3 h, and declined rapidly to resemble saline-treated controls at 6 h after. A 5.9-fold increase of FC was observed at 1 h with 5.0 mg/kg AMD 3100 (P = 0.012). These data suggest that AMD 3100 is a potent cell mobilizer from bone marrow to PB. We next investigated the effect of AMD 3100 in combination with FL and G-CSF on the mobilization of FC and HSC into PB. B6 mice were injected with FL (day 1 to 10), G-CSF (day 4 to 10), and AMD 3100 (day 10). PB was obtained 1 h after injection on day 10. After performing a count of peripheral WBC, cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−) and HSC (Lin−/Sca-1+/c-kit+) mobilization. The maximal mobilization of PBMC was observed when animals were treated with AMD 3100/FL/G-CSF. The numbers of PBMC with AMD3100/FL/G-CSF treatment increased with 17.2-fold and 6.4-fold when compared with controls treated with saline or AMD 3100 alone (P < 0.00001), respectively. A maximal elevation of both FC and HSC was detected when AMD 3100 was added to FL/G-CSF treatment and reached 1.91 ± 0.42 × 103/μl (Figure 1A) and 1.89 ± 0.35 × 103/μl (Figure 1B), respectively. The increase of FC and HSC was significant. There was a 10.1-fold increase in FC and 230.8-fold increase in HSC when compared with recipients treated with AMD 3100 alone (P < 0.00001). AMD 3100/FL/G-CSF treatment resulted in a 1.7-fold of FC and 2.2-fold increase of HSC when compared with recipients treated with FL/G-CSF (P < 0.05). In summary, AMD 3100, FL, and G-CSF show a highly significant synergy on the mobilization of FC and HSC. This study may be clinically relevant in efforts to mobilize immunomodulatory FC and HSC to PB for transplantation, especially to induce tolerance for organ transplant recipients. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

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Monoclonal Antibodies with Neuroblastoma Specificity: A Flow Cytometric Analysis of Cross-Reactivity with CD34+Hematopoietic Stem Cells

Journal of Hematotherapy & Stem Cell Research ◽

10.1089/152581600750062309 ◽

2000 ◽

Vol 9 (6) ◽

pp. 867-875 ◽

Cited By ~ 2

Author(s):

Marianne Ifversen ◽

Lisa D. Christensen ◽

Catherine Rechnitzer ◽

Carsten Heilmann

Keyword(s):

Stem Cells ◽

Monoclonal Antibodies ◽

Hematopoietic Stem Cells ◽

Flow Cytometric Analysis ◽

Cross Reactivity ◽

Hematopoietic Stem ◽

Flow Cytometric

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Flow cytometric assessment of lymphocyte subsets, lymphoid progenitors, and hematopoietic stem cells in allogeneic stem cell grafts

Bone Marrow Transplantation ◽

10.1038/sj.bmt.1703270 ◽

2001 ◽

Vol 28 (11) ◽

pp. 1073-1082 ◽

Cited By ~ 66

Author(s):

K Theilgaard-Mönch ◽

K Raaschou-Jensen ◽

H Palm ◽

K Schjødt ◽

C Heilmann ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Lymphocyte Subsets ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Lymphoid Progenitors

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Deletion of PTPN1 Promotes the Development of Myelofibrosis

Blood ◽

10.1182/blood.v128.22.795.795 ◽

2016 ◽

Vol 128 (22) ◽

pp. 795-795

Author(s):

Fatoumata Jobe ◽

Bhumika Patel ◽

Hideki Makishima ◽

Bartlomiej P Przychodzen ◽

Robert E Hutchison ◽

...

Keyword(s):

Stem Cells ◽

Tumor Suppressor ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Tyrosine Phosphatase ◽

Wild Type ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Obesity And Diabetes

Abstract Deletion of chromosome 20q [del(20q)] is a common chromosomal abnormality associated with myeloid neoplasms including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN overlap disorders and acute myeloid leukemia (AML). The del(20q) lesion is often associated with myeloproliferative features; it is present in patients with myelofibrosis (MF) at a high frequency (24%) and thus considered to be one of the most frequent cytogenetic abnormalities in MF (Wassie et al., Br J Haematol. 2015). The del(20q) lesion can also coexist with JAK2V617F mutation in MPN/MF. However, the target tumor-suppressor gene(s) within chromosome 20q involved in the pathogenesis of MF remains unknown. The PTPN1 locus maps to human chromosome 20q13.1-q13.2. PTPN1 (also known as PTP1B) is a ubiquitously expressed non-receptor tyrosine phosphatase that has been linked to metabolism and cancer. Mice deficient in Ptpn1 exhibit resistance to diet-induced obesity and diabetes. Both oncogenic and tumor suppressor functions for PTPN1 have been suggested. PTPN1 can negatively regulate the JAK/STAT signaling, which is frequently found activated in MPN. Here, we report the identification and functional consequences of PTPN1 deletion in the pathogenesis of MF. Deletion of PTPN1 was identified in 14% cases of MF. Conditional deletion of Ptpn1 in the mouse hematopoietic compartment resulted in significant increases in white blood cell and neutrophil counts in the peripheral blood and enlargement of spleen size. Flow cytometric analyses showed significant expansion of myeloid (Gr-1+/Mac-1+) precursors in the bone marrow (BM) and spleens of Ptpn1-deleted mice compared with control animals. Megakaryocytic (CD41+/CD61+) precursors were also significantly increased in the spleens of Ptpn1-deleted mice. Flow cytometric analyses also revealed significant increases in absolute numbers of LSK cells (Lin-Sca1+c-kit+) and its subsets including long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC) and multi-potent progenitors (MPP) in the spleens of Ptpn1-deleted mice. Hematopoietic progenitor colony assays showed significant increases in myeloid (CFU-GM) and megakaryocytic (CFU-Mk) colonies in the BM of Ptpn1-deleted mice compared with control mice BM. Histopathologic analysis demonstrated fibrosis (grade 2) in the BM and spleens of Ptpn1-deleted mice at 52 weeks after induction, whereas control animals did not exhibit fibrosis at that age. Together, these results suggest that deletion of Ptpn1 induces an MPN-like phenotype, which progresses to MF over time. Moreover, transplantation of Ptpn1-deficient BM into lethally irradiated wild-type animals resulted in fibrosis at 18 weeks after transplantation, demonstrating that the effect of Ptpn1 loss in the development of myelofibrosis is cell-intrinsic. Competitive repopulation assays using BM from control or Ptpn1-deficient (CD45.2+) mice with wild type congenic (CD45.1+) mice showed that deletion of Ptpn1 enhances the repopulation capacity of hematopoietic stem cells. Biochemical analyses revealed that depletion of Ptpn1 enhanced JAK2/STAT5, AKT and ERK signaling in the BM of Ptpn1-deleted mice. Furthermore, we observed that deletion of Ptpn1 in Jak2V617F knock-in mice accelerates the development of myelofibrosis. In conclusion, our results establish a tumor-suppressor function for PTPN1 in MF. Disclosures No relevant conflicts of interest to declare.

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