Functional CXCR4 Expressing Microparticles and SDF-1 Correlate with Circulating AML Cell Counts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4375-4375
Author(s):  
Alexander Kalinkovich ◽  
Sigal Tavor ◽  
Abraham Avigdor ◽  
Joy Kahn ◽  
Alexander Brill ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Proteolytic Enzymes ◽  
Cxcr4 Antagonist ◽  
Human Cd34 ◽  
Cell Counts ◽  
Normal Individuals ◽  
Bone Marrow Plasma ◽  
Stromal Cell Derived Factor

Abstract Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of AML. Cellular microparticles (MPs), the submicron vesicles shed from the plasma membrane of various circulating cells, are associated with numerous human disorders. In the present study, we studied the putative relationships between CXCR4/SDF-1 axis and MPs in AML. We detected CXCR4 expressing MPs (CXCR4+MPs) in the peripheral blood and bone marrow plasma samples of normal donors (n=24) and newly diagnosed adult AML patients (n=26). The majority of CXCR4+MPs in AML patients were CD45+ whereas in normal individuals they were mostly CD41+. In samples from AML patients, the levels of CXCR4+MPs and total SDF-1 were significantly elevated as compared to normal individuals. Importantly, we found a strong correlation between the levels of CXCR4+MP and white blood cell (WBC) counts in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, functional, non-cleaved SDF-1 levels were reduced in these patients compared to normal individuals, and also strongly correlated with the WBC counts. Furthermore, our data indicate N-terminal truncation of the CXCR4 molecule in the MPs of AML patients. This was found also in MPs obtained from the conditioned media of normal human CD34+ progenitors lentiviarlly transduced with CXCR4 vector in vitro. Appearance of MPs possessing N-terminally truncated CXCR4 in AML patients is likely to be dependent on proteolytic enzymes, such as elastase, which was elevated. However, MPs isolated from AML patients were capable of transferring functional CXCR4 molecule to the AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID mice. The CXCR4 antagonist AMD3100 reduced the increased migration and homing of MP treated HL-60 cells. Taken together, these findings suggest that functional CXCR4+MPs and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML parameter. Moreover, our findings suggest also the need for CXCR4- and SDF-1-target therapeutic approaches, clinically relevant in AML in the near future.

Evidence for An Anti-Cancer Barrier in a Mixed Lineage Leukemia Mouse Model in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3347-3347
Author(s):  
Sylvia Takacova ◽  
Jiri Bartek ◽  
Lucie Piterkova ◽  
Robert K. Slany ◽  
Vladimir Divoky
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Mouse Model ◽  
Peripheral Blood ◽  
Myeloid Cells ◽  
Mixed Lineage Leukemia ◽  
Cell Counts ◽  
Potential Tumor Suppressor

Abstract Mixed Lineage Leukemia (MLL) mutations identify a unique group of acute leukemias with distinct biological and clinical features. Although the role of MLL in leukemogenesis has been extensively studied, a precise mechanism regarding the leukemogenic potential of MLL mutations is not known. We generated a switchable MLL-ENL-ERtm mouse model, in which the MLL-ENL oncogene has been introduced by homologous recombination and is controlled by the endogenous MLL promoter, thus, expressed at physiological levels. Due to fusion with the estrogen receptor ligand binding domain (ERtm), the MLL-ENL-ERtm protein activity is dependent on continuous provision of tamoxifen or 4-hydroxytamoxifen. The MLL-ENL-ERtm mice have developed a myeloproliferative disorder (MPD) characterized by persistent mature neutrophilia after 484,5 +/− 75,68 days of latency on a tamoxifen diet, in association with high white cell counts in peripheral blood, splenomegaly and occasionally with anemia. Blood smears showed large numbers of mature myeloid elements consisting of 40–80% neutrophils (non-segmented forms in abundance), admixed with immature myeloid elements, 3–11% monocytes and 2–6% myeloblasts. The phenotype of MPD also involved myelomonocytic proliferation with 35% immature monocytic cells in one animal and severe anemia with increased numbers of immature erythroid cells in peripheral blood in another animal. Hematoxylin- and eosin-stained sections of the bone marrow from MLL-ENL-ERtm mice revealed expansion of myeloid cell population with no signs of progressive dysplasia. We observed massive infiltration of myeloid cells (positive for myeloperoxidase) into spleen with various degree of loss of normal splenic architecture depending on disease progression. FACS profiles of both bone marrow and spleen cells showed a typical pattern of granulocyte/macrophage/monocyte surface marker expression (CD34-CD43+Mac- 1+Gr-1+CD16/32+). In vitro evaluation of hematopoetic progenitors derived from bone marrow of leukemic mice at the terminal stage of the disease revealed decreased numbers of BFU-Es and increased numbers of CFU-GMs and CFU-Gs compared to matched controls. These results correlated with the expansion of the myelomonocytic and reduction of the erythroid compartment observed in the bone marrow of these animals. The average size (cellularity) of the mutant myeloid colonies was much smaller than the colonies derived from the wild-type controls, which could be caused by a partial block of terminal differentiation of myeloid progenitors in vitro. In vivo, MLL-ENL leads to expansion of differentiated myeloid cells in our model. High penetrance and long latency of leukemia in our model permits the study of early leukemia development. Our model revealed that MLL-ENL - induced myeloproliferation occurs as early as twelve weeks after MLL-ENL-ERtm activation in the bone marrow and infiltrates the spleen with a consequent decrease in lymphoid B220+CD19+IgM+ cells. Using the TUNEL assay on bone marrow sections, we observed induction of apoptosis in the highly proliferative bone marrow compartment compared to matched controls. These results suggest activation of a potential tumor suppressor mechanism by MLL-ENL in early stages of leukemia. We are currently investigating potential tumor suppressor pathways that might be involved in MLL-ENL - induced apoptosis in preleukemia.

scholarly journals An All Antibody Approach for Conditioning Bone Marrow for Hematopoietic Stem Cell Transplantation with Anti-cKIT and Anti-CD47 in Non-Human Primates

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4428-4428
Author(s):  
Kristopher D Marjon ◽  
James Y Chen ◽  
Jiaqi Duan ◽  
Timothy S Choi ◽  
Kavitha Sompalli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Mast Cell Degranulation ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership

Background Hematopoietic stem cell (HSC) transplantation (HSCT) is a well-established procedure that, with or without gene therapy, is curative for numerous severe life-threatening diseases including genetic blood disorders and blood cancers. While advances have been made, there are still substantial concerns since these chemo- and radiation therapy based procedures cause long-term toxicities such as infertility and secondary malignancies or even result in high mortality. We have previously established in a series of preclinical studies a novel chemo- and radiation-free non-toxic monoclonal antibody (Ab) -based conditioning regimen for autologous and allogeneic HSCT (Czechowicz et al., Akanksha et al. and George et al.). This cKIT-CD47 Ab-based regimen selectively depletes host HSCs for HSCT while sparing off-target toxicities caused by chemotherapy/radiation. By significantly decreasing morbidity/mortality associated with traditional conditioning regimens, antibody-mediated conditioning could expand the patient population eligible to receive HSCT for a variety of disorders. We developed a novel cKIT Ab (FSI-174), with an active Fc, and in combination with our CD47 magrolimab (previously 5F9, blocks the don't eat me pathway) could be utilized to translate the promising preclinical findings into clinical studies for safe and less toxic bone marrow conditioning for HSCT. Here we present the functional characterization of FSI-174 as single Ab and in combination with magrolimab in vitro and in non-human primate (NHP) studies. Methods We tested if FSI-174 could block stem cell factor signaling and we explored if FSI-174 alone or in combination with magrolimab could promote phagocytosis of cKIT positive cells (Kasumi-1). In addition, we determined if FSI-174 could cause mast cell degranulation. Subsequently, we explored the potential of FSI-174 alone (Phase A) or in combination with magrolimab (Phase B) to deplete HSCs in NHPs (rhesus macaques)in vivo. In Phase A, single doses of FSI-174 (0.3, 1, or 3 mg/kg) were administered alone. In Phase B, FSI-174 (0.3 or 3 mg/kg) was administered in combination with magrolimab (5mg/kg priming and 20 mg/kg maintenance dose). Bone marrow aspirates and core biopsies and peripheral blood were sampled before the study start and throughout the study. Frequency of bone marrow HSCs and cKIT receptor occupancy (RO) was determined by flow cytometry. In addition, the PK profile of FSI-174 was determined. Results In-vitro analysis demonstrated that FSI-174 decreases proliferation of HSPCs and enhances phagocytosis of cKIT positive cells, and the addition of magrolimab synergistically enhances the phagocytosis. Strikingly, FSI-174 did not cause mast cell degranulation in vitro. In the NHPs, complete (100%) cKIT receptor occupancy was achieved at all FSI-174 dose levels and was maintained for 1 to 9 days correlating with increasing doses and pharmacokinetics. The FSI-174 Cmax was found to be proportional to dose and mean Cmax increased from 6.25 ug/mL to 49.2 ug/mL. In Phase A, FSI-174 alone did not decrease the frequency of bone marrow HSCs compared to PBS control and had no effect on the peripheral blood cell counts. However, in Phase B, when FSI-174 was combined with magrolimab it significantly decreased the frequency of bone marrow HSCs with the nadir at day 9 and no recovery over 85 days compared to PBS control. Notably, there were no changes in peripheral blood cell counts over the course of the studies with no cytopenias in combination treatment. Conclusions We have developed a novel cKIT Ab (FSI-174) that meets the desired profile of stem cell factor block, promotion of phagocytosis, but without promoting mast cell degranulation. Furthermore, in the NHPs studies we have confirmed our chemo- and radiation-free cKIT-CD47 Ab -based conditioning approach with FSI-174 and magrolimab. As anticipated by our previous preclinical studies, monotherapy with FSI-174 does not deplete bone marrow HSCs in NHPs. Notably, no cytopenias are observed with either monotherapy or combination therapy. These data demonstrate the specificity, efficacy and safety of FSI-174/ magrolimab combination have great potential for conditioning regimen for HSCT in a chemotherapy and radiation free manner. Given the favorable safety profile of magrolimab across several clinical studies, these results are paving the way to the first-in-human trials for this novel conditioning for HSCT. Disclosures Marjon: Forty Seven Inc: Employment, Equity Ownership. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Feng:Forty Seven Inc: Employment, Equity Ownership. Mata:Forty Seven inc: Employment, Equity Ownership. Chen:Forty Seven Inc: Employment, Equity Ownership. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding; Kymab: Consultancy; Jazz: Research Funding. Chao:Forty Seven Inc: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Majeti:FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals The Chemokine SDF-1 Is a Chemoattractant for Human CD34+ Hematopoietic Progenitor Cells and Provides a New Mechanism to Explain the Mobilization of CD34+ Progenitors to Peripheral Blood

1997 ◽  
Vol 185 (1) ◽  
pp. 111-120 ◽  
Author(s):  
A. Aiuti ◽  
I.J. Webb ◽  
C. Bleul ◽  
T. Springer ◽  
J.C. Gutierrez-Ramos
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor ◽  
Transient Elevation

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38− or CD34+/DR−) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.

The CXCR4 Antagonist, AMD3100, Inhibits AML Transmigratory Activity but Does Not Alter Blast Proliferation or Survival.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2881-2881
Author(s):  
Jane L. Liesveld ◽  
Jeffrey E. Lancet ◽  
Karen E. Rosell ◽  
Jeremy Bechelli ◽  
Camille N. Abboud ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
In Vivo Studies ◽  
Cxcr4 Antagonist ◽  
Healthy Human ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor

Abstract Stromal cell derived factor-1 (SDF-1α) and its receptor, CXCR4 play a role in the trafficking of CD34+ cells. AMD3100, a selective CXCR4 antagonist, can mobilize hematopoietic progenitors from marrow to peripheral blood in healthy human volunteers and in patients with multiple myeloma and non-Hodgkin’s lymphoma (Flomenberg et al, Blood 102, 39a, 2003). Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation and NOD/SCID repopulating capacity (Kahn et al. Blood 103:2942, 2004). Since CXCR4 has been found to regulate the migration and development of AML stem cells in NOD/SCID mice, we studied the effect of AMD3100 on AML cells from the standpoint of proliferation and in vitro transendothelial transmigration utilizing a transwell system. AMD3100 (from AnorMED, Inc.), at concentrations from 0.1 to 1.0 ng/ml did not affect the viability or porliferation of purified AML blasts (n=4). AMD3100 did not influence the adherence of AML blasts to endothelial monolayers. In the presence of 0.1 to 1 ng/ml AMD-3100, the transmigration of normal CD34+ cells stimulated by 100 ng/ml SDF-1α through a human umbilical vein endothelial cell (HUVEC) monolayer was completely inhibited. Likewise, the transmigration of AML blasts through HUVECs was not altered by AMD3100 exposure, but the SDF-1α mediated transmigration was inhibited by AMD3100 from 0.1 to 1 ng/ml. The same effect was noted with AML transmigration through marrow stromal layers. The increase in transmigration through endothelial cells stimulated with G-CSF was not inhibited by AMD3100 whereas the transmigration stimulated by interleukin-8 was inhibited. When AMD3100 was placed in the bottom of the migration chamber, no independent effects on AML transmigration were noted. Co-culture of AML blasts with stromal monolayers protected blasts from apoptosis. This protection was not altered by SDF-1α, AMD3100, nor by the combination. These in vitro results demonstrate that AMD3100 can influence the migratory capacity of AML cells but has no direct effects on their proliferation or survival. Further in vitro and in vivo studies will be required to elucidate the role that this unique chemokine antagonist has in the mobilization potential of AML blasts or progenitors or in the interactions of AML cells with their microenvironment. Such studies have implications for AML autografting and AML blast interactions with extramedullary endothelial cells.

The sphingosine 1-phosphate receptor agonist FTY720 supports CXCR4-dependent migration and bone marrow homing of human CD34+ progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4478-4486 ◽  
Author(s):  
Takafumi Kimura ◽  
Andreas M. Boehmler ◽  
Gabriele Seitz ◽  
Selim Kuçi ◽  
Tina Wiesner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Actin Polymerization ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Sphingosine 1 Phosphate ◽  
Stem And Progenitor Cells ◽  
Stromal Cell Derived Factor

Abstract The novel immunosuppressant FTY720 activates sphingosine 1-phosphate receptors (S1PRs) that affect responsiveness of lymphocytes to chemokines such as stromal cell-derived factor 1 (SDF-1), resulting in increased lymphocyte homing to secondary lymphoid organs. Since SDF-1 and its receptor CXCR4 are also involved in bone marrow (BM) homing of hematopoietic stem and progenitor cells (HPCs), we analyzed expression of S1PRs and the influence of FTY720 on SDF-1/CXCR4-mediated effects in human HPCs. By reverse transcriptase-polymerase chain reaction (RT-PCR), S1PRs were expressed in mobilized CD34+ HPCs, particularly in primitive CD34+/CD38- cells. Incubation of HPCs with FTY720 resulted in prolonged SDF-1-induced calcium mobilization and actin polymerization, and substantially increased SDF-1-dependent in vitro transendothelial migration, without affecting VLA-4, VLA-5, and CXCR4 expression. In nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, the number of CD34+/CD38- cells that homed to the BM after 18 hours was significantly raised by pretreatment of animals and cells with FTY720, tending to result in improved engraftment. In addition, in vitro growth of HPCs (week-5 cobblestone area-forming cells [CAFCs]) was 2.4-fold increased. We conclude that activation of S1PRs by FTY720 increases CXCR4 function in HPCs both in vitro and in vivo, supporting homing and proliferation of HPCs. In the hematopoietic microenvironment, S1PRs are involved in migration and maintenance of HPCs by modulating the effects of SDF-1. (Blood. 2004;103:4478-4486)

scholarly journals Generation of Synthetic T-ALL By De Novo Transformation of Human Cord Blood Progenitors with a 4-Oncogene Cocktail

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3675-3675
Author(s):  
Manabu Kusakabe ◽  
Claire Shanna ◽  
Xuehai Wang ◽  
Catherine Jenkins ◽  
Vincenzo Giambra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Human Cells ◽  
Post Transplant ◽  
Cell Counts ◽  
Flow Cytometric

Abstract Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of blood cancer that can arise in both children and adults. Numerous studies have explored the effects of putative T-ALL oncogenes in mouse models and have contributed significantly to our understanding of disease pathogenesis. Nonetheless, it is clear there are important differences between mouse and human cells, particularly with respect to cellular transformation, and additional work is therefore needed to generate more accurate models of human disease. We sought here to create human T-ALL in the lab from normal CB progenitors by lentiviral transduction with a combination of known T-ALL oncogenes. Methods: Human CD34+ hematopoietic progenitor cells were isolated from pooled cord blood by magnetic bead/flow cytometric sorting (MACS/FACS). Sorted cells were then transduced by lentiviral vectors encoding a combination of four known T-ALL oncogenes including activated NOTCH1. NOTCH1 virus was marked with a GFP reporter (N1/GFP) while the other three accessory oncogenes were marked with a Cherry reporter (3xOnc/Cherry). Transduced cells were cultured on OP9-DL1 stromal feeders briefly prior to transplantation into NOD/SCID-IL2Rg-null (NSG) mice to assess leukemogenesis, or for longer periods to study their behavior in vitro. Results: Initial transduction efficiencies were typically 3-5% for each virus with 1-2% doubly-transduced N1/GFP+, 3xOnc/Cherry+ cells (hereafter referred as 4xOnc cells). After 28 days culture in vitro, the 4xOnc population reproducibly expanded and outcompeted singly- and non-transduced populations, accounting for more than 70% of cells in mixed cultures. By absolute cell counts, non-transduced cells stopped expanding within the first few weeks; however, 4xOnc cells kept expanding even after 6 weeks of culture. To test leukemogenesis in vivo, CD45+ cells were FACS sorted after 10 days of culture on OP9-DL1 feeders (including doubly-, singly-, and non-transduced populations) and injected intrahepatically into NSG neonates. Engraftment of human cells was followed monthly by flow cytometry of peripheral blood. Engraftment of GFP+ Cherry+ 4xOnc cells was first detected 2 months after transplantation whereas no engraftment of singly- or non-transduced cells was detected. The level of engraftment was below 5% and did not increase substantially even after 6 months following transplantation. At day 203 post-transplant, the primary recipient was sacrificed and 4xOnc cells were recovered from bone marrow, spleen and thymus where the levels of engraftment were approximately 10%. 4xOnc cells from the primary recipient were then serially transplanted into secondary recipients. Engraftment of 4xOnc cells in secondary recipients was observed 5 weeks after transplant. Unlike the primary recipient, however, the percentage of 4xOnc cells in the peripheral blood of secondary recipients gradually increased and these animals developed clinically morbid disease by 20 weeks post-transplant. At the time of necropsy, splenomegaly, lymphadenopathy, and enlarged thymus were observed and the bone marrow contained 80-90% 4xOnc cells. By flow cytometric analyses, 4xOnc cells expressed CD2, CD3, CD7, CD38, and TdT supporting acute T-cell leukemia. Also, TCR gamma clonality assay was performed with genomic DNA from 4xOnc cells from secondary recipients and revealed of 5-7 distinct clonal populations. These in vitro and in vivo findings were observed with multiple experimental replicates and with different pools of cord blood. Conclusion: Our in vitro and in vivo results suggest that NOTCH1, in combination with 3 accessory oncogenes are sufficient to transform normal human blood cells into clonal T-ALL-like malignant cells. Although we cannot exclude the possibility of the spontaneous acquisition of additional co-operating genetic or epigenetic abnormalities, this model provides a significant step forward to reveal the mechanisms involved in human T-ALL pathogenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

Evaluation of Congenital Neutropenic Disorders by in Vitro Bone Marrow Culture

PEDIATRICS ◽  
1977 ◽  
Vol 59 (5) ◽  
pp. 739-748
Author(s):  
Peter M. Falk ◽  
Kenneth Rich ◽  
Stephen Feig ◽  
E. Richard Stiehm ◽  
David W. Golde ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Severe Disease ◽  
Stem Cell Differentiation ◽  
Peripheral Blood Cell ◽  
Clinical Expression ◽  
Essentially Normal ◽  
Cell Counts

The congenital neutropenias are a heterogeneous group of diseases whose etiology and pathogenesis are largely unknown. We studied nine neutropenic patients from seven families. Evaluation included peripheral blood cell and differential cell counts, epinephrine and typhoid vaccine stimulation studies, Rebuck skin windows, and bone marrow aspirations for morphological assessment and for in vitro culture in liquid suspension and in agar plates. Parallel cultures were set up with and without colony-stimulating activity (CSA), and peripheral leukocytes were assayed for cellular production of CSA. Patients were initially classified on the basis of their clinical course: benign, mild, moderately severe, or severe disease. One patient in the moderately severe group had an immunoglobulin disorder. Morphologically normal mature granulocytes were seen in bone marrow aspirates of two patients, and maturational defects of varying degree were seen in the remaining seven. Colony formation in agar was markedly reduced below normal in three of seven, moderately reduced in two of seven, and greater than normal in two patients. Colonies in six of seven patients consisted exclusively of macrophages. Marrow from all but one of the nine patients demonstrated poor neutrophil development in suspension culture, and addition of CSA did not result in augmented granulocytic proliferation or maturation. A scheme of normal neutrophil maturation is proposed, and the nine patients were categorized according to this scheme. Four patterns of congenital neutropenia emerged: type 1 was the most benign form of disease with essentially normal clinical and in vitro parameters, and a defect considered to be due to a small committed stem cell pool, abnormal release, or excessive utilization peripherally; type 2 had mild disease with presumed defective committed stem cell differentiation along the granulocyte line; type 3 included benign to severe clinical expression with an apparent defect at the level of the committed granulocyte precursor more severe than in type 2; type 4 disease had varied clinical expression but evidence for a defect at the level of the pluripotent stem cell.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

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