Flow Cytometric Analysis of Signaling and Apoptosis in Hematopoietic Stem Cells

2014 ◽  
pp. 79-87 ◽  
Author(s):  
Lei Dong ◽  
Cheng-Kui Qu
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Flow Cytometric

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

Blood ◽  
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.

High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 855-862 ◽  
Author(s):  
Robert A. J. Oostendorp ◽  
Julie Audet ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Flow Cytometric ◽  
Differentiation Status ◽  
Kinetics Of

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint-Apoptosis Coupling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3357-3357
Author(s):  
Sara Rohrabaugh ◽  
Charlie Mantel ◽  
Hal E. Broxmeyer
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Mitotic Spindle ◽  
Flow Cytometric Analysis ◽  
Spindle Checkpoint ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Mitotic Spindle Checkpoint

Abstract Cell cycle checkpoints guarantee that cells move through the events of the cell cycle in the appropriate manner. The mitotic spindle checkpoint, also known as the spindle assembly checkpoint (SAC), helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Our lab recently reported on the condition of the SAC in both mouse and human embryonic stem cells (ESCs). We found that ESCs do not initiate apoptosis when the SAC is activated, which allowed these cells to tolerate a polyploid state resulting from the aberrant mitosis (Mantel et al. Blood.109: 4518–4527. 2007). These results lead us to conclude that the spindle checkpoint is uncoupled from apoptosis in ESCs. Knowing whether adult tissue specific stem/progenitor cells, such as hematopoietic stem cells (HSCs), have checkpoints which are uncoupled from apoptosis is extremely important information. If HSCs were to manifest such checkpoint uncoupling as that which we defined for ESCs, this might present a problem for the ex-vivo expansion and transplantation of HSCs. Using multiparametric permeablized cell flow cytometric analysis, we found the mitotic spindle checkpoint to be functional in primary murine sca 1+/c-kit+/lin- cells (LSK cells), a population highly enriched in primitive hematopoietic stem/progenitor cells. Using nocodazole, which exerts its affect by depolymerizing microtubules, we were able to activate the spindle checkpoint in low density mononuclear cells collected from murine bone marrow. Through flow cytometric analysis of the LSK cells in the mononuclear fraction, we were able to determine that spindle checkpoint activation in LSK cells resulted in a cell cycle arrest in mitosis, which was determined by DNA content of the cells, and eventually this arrest lead to cell death via apoptosis, as indicated by caspase-3 activation. This behavior is unlike that of ESCs, which exit mitosis and become polyploidy after prolonged spindle checkpoint activation. Thus the mitotic spindle checkpoint appears to be coupled to apoptosis in this particular set of tissue specific stem/progenitor cells, which lessens the possibility that ex-vivo expansion of hematopoietic stem cells will result in abnormalities to these cells that may give rise to disease initiation or progression after their transplantation.

Targeting Acute Myeloid Leukemia Stem Cells by MUC1-C Subunit Inhibition

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 848-848 ◽  
Author(s):  
Dina Stroopinsky ◽  
Jacalyn Rosenblatt ◽  
Keisuke Ito ◽  
Li Yin ◽  
Hasan Rajabi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Control Peptide ◽  
Cd34 Cells ◽  
Acute Myeloid

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.

scholarly journals Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1706-1712 ◽  
Author(s):  
S Okada ◽  
H Nakauchi ◽  
K Nagayoshi ◽  
S Nishikawa ◽  
S Nishikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Tyrosine Kinase Receptor ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Marrow Cells

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK- 2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr- 1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c- kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

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

2001 ◽  
Vol 28 (11) ◽  
pp. 1073-1082 ◽  
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

The Identification and Characterization of Zebrafish Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 681-681
Author(s):  
Dongdong Ma ◽  
Hui-feng Lin ◽  
Robert Irving Handin
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Flow Cytometric Analysis ◽  
Great Promise ◽  
Transgenic Zebrafish ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and maintain hematopoiesis throughout the lifespan of an organism. Because of the optical clarity of their embryos and the ease of genetic manipulation, the zebrafish (Danio rario) is an excellent model for studying hematopoiesis. To date, though, there have been no published studies characterizing zebrafish HSCs. We have engineered transgenic zebrafish strains with green fluorescent protein (GFP+) thrombocytes by driving expression of the GFP reporter with the platelet/thrombocyte-specific CD41 promoter (Lin et. al. Blood. 2005 Dec 1; 106: 3803). We have identified two populations of CD41-GFP+ cells in suspensions of whole kidney marrow by flow cytometry. The majority of the cells strongly express GFP (GFPhi) and are mature thrombocytes. There is also a small population of immature cells, which express GFP weakly (GFPlo), that are of uncertain lineage. Peripheral blood and splenic cell suspensions contain only GFPhi cells. Studies in humans and mice have shown that CD41 is transiently expressed on the earliest hematopoietic progenitors and is then silenced, suggesting that the GFPlo cells might be hematopoietic progenitors or HSCs. To test this hypothesis, we transplanted flow-sorted GFPhi and GFPlo cells into gamma-irradiated adult AB strain zebrafish. The GFPhi cells were no longer detected 7 days after transplantation. In contrast, we have observed long-term engraftment and multi-lineage reconstitution for over 6 months after transplantation of GFPlo cells into zebrafish conditioned with gamma irradiation at 25 Gray or 40 Gray. Multi-lineage reconstitution persisted following serial transplantation of the GFPlo cells. Initial transplants were carried out with 105 GFPlo cells. To test the self renewal properties of GFPlo cells, transplants were carried out with injections of 1000 to 1 GFPlo cell(s)/fish. Multi-lineage reconstitution was seen with all doses and was achieved in 5 of 54 fish after transplantation of a single GFPlo cell. Flow cytometric analysis of whole kidney marrow from fish examined six months after transplantation demonstrated the persistence of GFPlo cells. Taken together, these studies suggest that GFPlo cells are capable of long-term hematopoietic reconstitution and are true HSCs. We believe that the zebrafish HSC transplant model holds great promise for the in vivo study of HSCs and may provide a valuable tool to track HSC homing, proliferation and differentiation.

Chronic RPS19 Deficiency Leads to Bone Marrow Failure In a Mouse Model for Diamond-Blackfan Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 193-193
Author(s):  
Pekka Jaako ◽  
Johan Flygare ◽  
Karin Olsson ◽  
Ronan Quere ◽  
Jonas Larsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Flow Cytometric

Abstract Abstract 193 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al, 2009 ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the Collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells, and flow cytometric analysis of bone marrow after a short-term induction reveals increased frequencies of hematopoietic stem and progenitor cells reflecting the onset of stress hematopoiesis. In the current study we have analyzed the long-term effect of RPS19 deficiency in bone marrow. In contrast to a short-term induction, flow cytometric analysis of bone marrow after 51 days revealed decreased frequencies of hematopoietic stem and progenitor cells that correlate with a severe peripheral blood phenotype. In addition, we observed a 3–6 fold increase in apoptosis in RPS19-deficient bone marrow compared to controls based on TUNEL assay. Furthermore, transplantation of whole bone marrow cells from transgenic donors into wild type lethally irradiated recipients confirms that the observed phenotype is autonomous to the blood system. To study whether long-term RPS19 deficiency functionally impairs hematopoietic stem cells, we pre-induced mice for 30 days followed by 15 days without doxycycline to restore the RPS19 expression. Mice were sacrificed and total bone marrow cells were transplanted together with wild-type competitor cells (1:1) into wild type lethally irradiated recipients without doxycycline. This experimental setting allows us to assess the functionality of pre-induced hematopoietic stem cells in absence of ribosomal stress. Flow cytometric analysis of peripheral blood one month after transplantation clearly demonstrates decreased reconstitution from pre-induced donors compared to the wild-type competitor. While this time point reflects mainly the function of transplanted progenitors, long-term analysis of hematopoietic stem cell function in these recipients is ongoing. To study the molecular mechanisms underlying the hematopoietic defect we performed comparative microarray analysis. We chose to analyze preCFU-E/CFU-E erythroid progenitors since we have previously located the erythroid defect at the CFU-E – proerythroblast transition based on flow cytometry and clonogenic proliferation cultures of prospectively isolated erythroid progenitors. Microarray analysis of preCFU-E/CFU-E progenitors reveals deregulation of several genetic pathways, including a robust upregulation of p53 pathway genes, and these targets have been confirmed by real-time PCR. Furthermore, many of p53 target genes are also upregulated in the Lineage− Sca-1+ c-Kit+ (LSK) population that contains immature hematopoietic progenitors and stem cells suggesting that the activation of p53 is not restricted to the erythroid lineage. To ask whether increased activity of p53 can solely explain the hematopoietic phenotype, we have crossed our mouse model into a p53-null background. In summary, our data suggest that RPS19-deficient mice fail to uphold stress hematopoiesis for extended periods of time, with chronic RPS19 deficiency causing bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

Deletion of PTPN1 Promotes the Development of Myelofibrosis

Blood ◽  
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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