Novel Methods to Quantitate and Identity Dividing Platelets in Culture Conditions

Abstract Background: Unlike dividing cells, platelets are usually regarded as unproductive in that they are anuclear fragments of megakaryocyte and incapable of the canonical cell cycle. However, recent evidence showed that platelets could form progeny or at least undergo fission. The observation indicated the existence of a non-hematopoietic mechanism to determine platelet count in peripheral blood. The assessing the kinetic aspects of platelet division is not easy because automated cell counters often give equivocal results after culture not supporting those previous findings. Manual chamber counting is impractical because of the difficulty in differentiating singlets from any small sized clumps. Objective: We examined platelet division under in-vitro culture conditions to assess how many platelets and how fast they divide. Methods: We devised a method to track platelet division based on flow cytometry and fluorescent microscopic image. Briefly, buffered human platelet suspension was prepared and stained with cell tracking dyes followed by sorting and resuspension in cell culture medium and incubation. Platelet division was assessed quantitatively by observing a decrease in fluorescent intensity by flow cytometry after culture in a liquid medium or counting the number of single colored platelet doublets after culture in hydrogel medium. Results: After 6-hour culture of CFSE labelled platelet in M199 medium, we observed the appearance of a separate cluster of platelets with reduced CFSE intensity adjacent to the original platelet cluster, as is the typical finding of CFSE dilution assay for lymphocyte proliferation. We continued the culture for 20 hours. The division fraction of strongly labeled platelets was 48.7 % (range: 27.5 - 70.4) after 6 hours and 85.6 % (79.1 - 92.0) after 20 hours. For weakly labelled platelets, the division fraction after 6 hours was 35.0 % (24.6 - 43.4) and 75.5 % (34.5 - 97.0) after 20 hours. The division fractions at the two time points were much higher than expected and the effect of external stimuli such as shearing force exposure during the sorting procedure was to be considered. In another assay, we divided platelet suspension into two and stained with labelling dyes of different colors (CFSE and FarRed). The two fractions were mixed and cultured in hydrogel medium. At the beginning of the culture and after 6 hours, we counted platelet singlets and doublets in ten randomly selected fields of con focal microscopy. Single colored doublets can form both by division and contact. Those with two colors can form only by contact. The count of single- and two-colored doublets can be estimated by binary probability prediction if the platelets don't divide. Initially, the doublet counts follow the binary probability prediction (P: 0.65 - 0.88), but after 6 hours, single colored doublets were observed more frequently than two colored doublets (P<0.01, in the order of 10-6). The division was suppressed by treatment with taxol (P =0.26), nocodazole (P=0.38), and cytochalasin D (P=0.61). From the counted numbers of doublets and singlets of the two colors, we could derive a formula for the platelet division fraction. The division fraction of FarRed stained platelets were 8.3 % (95% CI: 2.2 - 14.4), and that of the CFSE stained platelets were 11.9 % (5.4 - 18.4). Conclusion: We introduce novel methods to assess platelet division credibly in culture conditions which can be easily combined with functional studies. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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Preclinical Activity of Brentuximab Vedotin (SGN-35) in Primary Effusion Lymphoma (PEL),

Blood ◽

10.1182/blood.v118.21.3728.3728 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3728-3728 ◽

Cited By ~ 1

Author(s):

Shruti Bhatt ◽

Brittany Ashlock ◽

Yaso Natkunam ◽

Juan Carlos Ramos ◽

Enrique Mesri ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Conflicts Of Interest ◽

Primary Effusion Lymphoma ◽

Large Cell ◽

Brentuximab Vedotin ◽

Antibody Drug Conjugate ◽

Human Herpes Virus 8

Abstract Abstract 3728 Primary effusion lymphoma (PEL) is a distinct and aggressive subtype of non-Hodgkin lymphoma (NHL) commonly presenting with pleural, peritoneal, or pericardial malignant effusions usually without a contiguous tumor mass. PEL is most commonly diagnosed in HIV-positive patients, accounting for 4% of all NHLs in this population, yet may also develop in immunosuppressed HIV-negative individuals. While Human Herpes Virus 8 (HHV8 or Kaposi's sarcoma-associated herpesvirus) is directly implicated in the oncogenesis of this lymphoma, most PEL cases are also associated with Epstein-Barr virus and the combination of the two may facilitate transformation. The tumor cells exhibit plasmablastic features and express CD45, CD38, CD138, HHV8 and CD30. PEL is an aggressive tumor characterized by a short median survival of only 6 months with current therapeutic approaches underscoring the urgent need for development of new therapeutics. Brentuximab vedotin (SGN-35) is an antibody-drug conjugate (ADC) comprised of an anti-CD30 monoclonal antibody cAC10 conjugated by a protease-cleavable dipeptide linker to a potent cell killing agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and is transported to lysosomes, where the peptide linker is selectively cleaved allowing binding of the released MMAE to tubulin and leading to cell cycle arrest and apoptosis. Brentuximab vedotin was recently reported to have promising antitumor activity in CD30 expressing tumors, such as Hodgkin and Anaplastic large cell lymphomas. Since PEL tumors are reported to express CD30, we have hypothesized that brentuximab vedotin might be effective in the treatment of this NHL subtype. Initially, we have confirmed by flow cytometry the expression of CD30 on PEL cell lines (UM-PEL 1, UM-PEL 3, BC-1 and BC-3), and by review of immunohistochemistry and flow cytometry results in patients with previous diagnosis of PEL at our institution. To examine in vitro potency of brentuximab vedotin, UM-PEL 1, UM-PEL 3, BC-1 and BC-3 PEL cell lines were treated with brentuximab vedotin at concentration ranging from 0–100 micrograms/ml. Staining with YO-PRO and Propidium Iodide (PI) demonstrated dose dependent cell apoptosis and death in all the cell lines at 72 hours post treatment. In contrast, control IgG conjugated with MMAE failed to induce apoptosis and cell death of PEL cell lines confirming specific brentuximab vedotin cytotoxicity. Furthermore, brentuximab vedotin decreased proliferation of PEL cells at 48 hours leading to a complete proliferation arrest at 72 hours, as measured by MTS assay. These effects were absent after equivalent doses of control IgG conjugated drug treatment. Supportive to this, labeling of cells with PI to detect active DNA content by flow cytometry showed that bretuximab vedotin induced growth arrest in G2/M phase. To further establish the anti-tumor potential of brentuximab vedotin in vivo, we used the direct xenograft UM-PEL 1 model, established in our laboratory (Sarosiek, PNAS 2010), which mimics human PEL tumors. UM-PEL 1 bearing mice were injected intraperitoneally 3 times a week with brentuximab vedotin or control IgG conjugated MMAE for 4 weeks. Brentuximab vedotin treatment markedly prolonged overall survival of UM-PEL-1 bearing mice compared to controls (p Disclosures: No relevant conflicts of interest to declare.

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Plasmin Cleaves Complement iC3b, Preventing CR3/4 Mediated Phagocytosis By Macrophages

Blood ◽

10.1182/blood.v122.21.1105.1105 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1105-1105

Author(s):

Erica A. Peterson ◽

Jonathan H Foley ◽

Michael J Krisinger ◽

Edward Conway

Keyword(s):

Flow Cytometry ◽

Inflammatory Responses ◽

Conflicts Of Interest ◽

Tissue Injury ◽

Complement Receptors ◽

Red Cell Membrane ◽

Western Blots ◽

Complement Pathways

Abstract Introduction The plasmin(ogen) and complement systems are activated at sites of tissue injury and are involved in hemostasis, wound healing, inflammation and immune surveillance. Although the mechanisms are poorly understood, dysregulation of these systems underlie the pathogenesis and progression of inflammatory and vascular diseases. We aimed to characterize the relevant molecular interactions between the plasmin(ogen) and complement pathways. The three complement pathways converge with formation of C3-convertases that cleave C3 into C3a and C3b. C3a is liberated as an anaphylatoxin while C3b participates in further formation of the C3 and C5 convertases, thereby amplifying complement activation. To dampen the system, negative regulatory mechanisms exist. C3b is degraded to iC3b by the factor I (FI)/FH complex, which in turn is degraded to C3dg by the FI/complement receptor 1 (CR1) complex. iC3b and C3dg induce cellular responses by binding to complement receptors CR3 / CR4 / CR2, and CR2, respectively. Interactions of iC3b with CR3 or CR4 induce phagocytosis by macrophages, and binding of iC3b or C3dg to CR2 promotes B-cell responses. Recent studies show that plasmin proteolyses C3b and iC3b. We further characterized the plasmin cleavage sites in iC3b and evaluated the functional consequences in vitro. Methods and Results Plasmin cleavage of iC3b was examined over a range of concentrations and times. Plasmin (50 nM) generated a 40 kDa iC3b cleavage fragment (946TLD – PSR1303) which was notable for containing both C3dg (1002HLI – PSR1303) and the C3 thioester domain, necessary for opsonic binding to surfaces. We tested the relevance of this cleavage in phagocytosis assays using immunofluorescence and flow cytometry (Figure 1). C3b bound to the surface of fluorescent (Alexa 488) zymosan particles (C3b-zym), was treated with FI/FH to generate iC3b-zym, and subsequently incubated with FI/CR1 or plasmin to yield C3dg-zym or 946TLD – PSR1303-zym, respectively. Western blots confirmed that plasmin generated 946TLD – PSR1303 from iC3b-zym. The C3 fragment-zymosan species (C3b-zym, iC3b-zym, C3dg-zym and 946TLD – PSR1303-zym) were each incubated with macrophages (PMA-differentiated THP-1 cells) for 90 minutes. Cells were washed, stained and fixed for immunofluorescence, or suspended for flow cytometry. Figure 1, panel A shows macrophages stained with CellMask (red, cell membrane) and DAPI (blue, nucleus). Fluorescent zymosan is seen in green. No phagocytosis was detected with zymosan lacking C3 (zym alone), but there was a small amount with C3b-zym. In contrast, iC3b-zym was highly effective in inducing phagocytosis by most macrophages. This effect of iC3b-zym was abolished with FI/CR1 or plasmin, i.e. little phagocytosis was detected with C3dg-zym or 946TLD – PSR1303-zym. Flow cytometry-based quantitative analyses confirmed the preceding findings (Figure 1, panel B), with a similar pattern of phagocytosis induced by the zymosan-bound fragments. No phagocytosis was detected with zymosan lacking C3. Phagocytosis of C3b-zym and iC3b-zym was 7±2% and 17±1% of cells, respectively. C3dg-zym and 946TLD – PSR1303-zym induced phagocytosis was <5%. We also evaluated the role of the complement receptors in mediating the effect of the C3b/iC3b fragments using CR3/4 and CR1 blocking antibodies. These confirmed that phagocytosis of iC3b-zym and C3b-zym is mediated by CR3/4 and CR1, respectively. Conclusions Plasmin cleaves iC3b to form a redundant complement regulatory pathway with the FI/CR1 complex, but which notably does not require a cellular cofactor. Further studies will delineate the role of this and other plasmin-generated complement fragments in modulating innate immune and inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

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Post-Transplant Immune Monitoring In Patients Receiving Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.5486.5486 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5486-5486

Author(s):

Silvia Park ◽

Chul Won Jung ◽

Jun Ho Jang ◽

Eun Suk Kang ◽

Kihyun Kim

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Conflicts Of Interest ◽

Immune Monitoring ◽

Disease Relapse ◽

Cell Secretion ◽

Color Flow ◽

Blood Samples ◽

Post Transplant

Abstract Introduction There are still substantial morbidity and mortality caused by insufficient immunologic recovery after allo-HSCT. In this context, we attempt to evaluate the clinical relevance of immune monitoring in allo-HSCT recipients. Method Fifty five patients who underwent allo-HSCT between 2008 and 2012 were included. Peripheral blood samples were drawn from recipients before transplant, and on 4, 8, 12, 24, 36 and 48 weeks after transplant. Each blood samples were analyzed by multi-color flow cytometry for determining lymphocyte subsets. MNC were separated from blood specimen, and analyzed for the quantitation of Treg with the use of real-time PCR. We also examined T cell derived IFN-r by using in vitro culture, intracellular staining, and flow cytometry analysis. Results The median age was 43, and AML was the most common reason for transplantation (49.1%). Grade II or more aGVHD occurred in 36.4% of cases, and 49.1% exhibited moderate or severe cGVHD. The differences in the proportion (%) and the absolute number (/uL) of CD4+, CD8+ cells, CD4+ derived IFN-r (%), CD8+ derived IFN-r (%), and Treg (%) between the groups (Gr. II or more aGVHD (+) vs (-); moderate or severe cGVHD (+) vs (-)) were compared by Two sample t-test. Patients with Gr. II or more aGVHD showed decreased CD4+ count at 4, 8 and 12 weeks, but showed rather higher CD8+ count at 8 weeks after transplant. T-cell secretion function assessed by IFN-r (%), and Treg (%) was similar between two groups within 12 weeks after transplant. In case of cGVHD, both CD4+ and CD8+ count tended to be higher in patients with moderate or severe cGVHD, and the trends lasted for up to 48 weeks from allo-HSCT. Treg (%) was almost consistently lower throughout the period in these patients. There were 12 relapses within follow up period (median 36.1 months), and higher slope of post-transplant increase in CD8+ count and CD8 derived IFN-r were identified as protective factors for disease relapse. Conclusion In view of the results so far achieved, slow recovery of CD8 count and function might be associated with disease relapse. However, this is still a preliminary data, and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

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Inflammation Response Cytokines IFN-γ and IL-10 Regulate Monocyte Subset Differentiation

Blood ◽

10.1182/blood-2019-129515 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3586-3586

Author(s):

Hui Zhong ◽

Weili Bao ◽

Yunfeng Liu ◽

Karina Yazdanbakhsh

Keyword(s):

Flow Cytometry ◽

Inflammatory Response ◽

Inflammatory Disease ◽

Disease Risk ◽

Conflicts Of Interest ◽

Response To Treatment ◽

Monocyte Subset ◽

Monocyte Differentiation ◽

Ifn Γ

Circulating monocytes comprise of a heterogeneous and functionally-diverse cell population which based on surface markers can be divided into three subsets: classical (CMo), intermediate (IMo), and non-classical monocytes/patrolling monocytes (PMo). The frequency/number, gene expression profile and activity of IMo and PMo significantly change in a variety of inflammatory diseases with the changes associated with disease risk and severity as well as response to treatment. While it is believed that CMo differentiate into IMo and that IMo further differentiate into PMo, there is paucity of data on the mechanisms that alter CMo to IMo/PMo differentiation profiles in these conditions. In addition, factors which induce human and mouse IMo and PMo differentiation have yet to be identified. To screen cytokine/chemokine candidates affecting IMo/PMo differentiation, human monocytes were isolated from healthy donors (HD) and cultured with candidates (22 cytokines/chemokines) for 3 days. On day 3, IMo/PMo marker expression was examined by flow cytometry focusing on candidate molecules that led to increased expression of markers (CD16, CX3CR1, CD11c, HO-1, HLA-DR) whose levels are normally found to be higher in IMo/PMo and to decrease in expression of markers (CD14, CD36, CCR2) which are expressed at higher level in CMo as measured by mean fluorescence intension (MFI). We found that of all molecules tested, only two , IFN-γ and IL-10, had significant effects: IFN-γ (10ng/ml) increased expression of CX3CR1 (20 fold), CD16 (60%), HLADR (15%) and inhibited CD36 (42% inhibition) and CD14 expression (45% inhibition) while IL-10 (10ng/ml) increased CD16 (3.3 fold), CD11c (45%), HO-1 (59%) and CX3CR1 (6 fold) expression and inhibited CCR2 (60% inhibition) expression. These data suggest that IFN-γ and IL-10, two key cytokines involved in sterile and infectious inflammation, induce IMo and PMo differentiation. To test whether the effect of IFN-γ and IL-10 in human in vitro cultures can be replicated in vivo, wildtype B6 mice were I.V. injected with IFN-γ or IL-10 for 3 days: IFN-γ, IL-10, or the same volume of PBS. The frequencies of monocyte subsets in blood (gated on CD45+Ly6G-CD11bhighCD115+ for total monocyte population and CMo/IMo/PMo based on Ly6C expression level) on day 4 were analyzed by flow cytometry. We found that IFN-γ (2.5μg/injection/mice twice/day) significantly increased IMo frequencies (from 12% to 35%) but decreased PMo frequencies (from 38% to 26%) while IL-10 (0.25μg/injection/mice twice/day) significantly induced PMo differentiation (from 38% to 63%) without effect on IMo frequencies. The data suggest that IFN-γ increases IMo frequency by simultaneously inducing CMo differentiation into IMo and inhibiting IMo differentiation into PMo. We have previously reported lower PMo frequency in patients with sickle cell disease (SCD), considered an inflammatory disease with altered immune profiles. To test whether altered differentiation programming of IMo/PMo may contribute to reduced PMo frequency in SCD, we analyzed the frequency of CMo/IMo/PMo at baseline and after IFN-γ or IL-10 injection to mimic an inflammatory response in AA mice (expressing normal human hemoglobin) and SS mice (expressing human SCD hemoglobin). We found significantly lower IMo frequency before treatment (AA vs SS:15.0% vs 9.2%) but also lower induction of IMo following IFN-γ treatment in SS mice (18%) relative to AA mice (35%), suggesting that IFN-γ inhibition of IMo differentiation into PMo in SCD is impaired. Furthermore, IL-10 was less effective in inducing PMo in SS as compared to AA mice (SS vs AA: 40% vs 60%). These data suggest that IFN-γ or IL-10-mediated monocyte differentiation in SCD is altered. Altogether, these data have unraveled a novel role for IFN-γ or IL-10, two key cytokines known to be induced during an inflammatory response, in monocyte differentiation, and suggest that IMo/PMo differentiation in a chronic inflammatory disease such as SCD may be defective due to altered response to IFN-γ and IL-10, opening up the potential for identification of novel therapeutic targets for IMo/PMo associated diseases including SCD. Disclosures No relevant conflicts of interest to declare.

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Latent tri-lineage potential of human menstrual blood-derived mesenchymal stromal cells revealed by specific in vitro culture conditions

10.21203/rs.3.rs-290653/v1 ◽

2021 ◽

Author(s):

Diana Quintero-Espinosa ◽

Viviana Soto-Mercado ◽

Catherine Quintero-Quinchia ◽

Miguel Mendivil-Perez ◽

Carlos Velez-Pardo ◽

...

Keyword(s):

Flow Cytometry ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Culture Conditions ◽

Menstrual Blood ◽

Neurologic Disorders ◽

Cellular Source ◽

Timely Fashion ◽

Direct Transdifferentiation

Abstract Human menstrual blood-derived mesenchymal stromal cells (MenSCs) have become not only an important source of stromal cells for cell therapy but also a cellular source for neurologic disorders in vitro modeling. By using culture protocols originally developed in our laboratory, we show that MenSCs can be converted into floating neurospheres (NSs) using the Fast-N-Spheres medium for 24-72h, and can be transdifferentiated into functional dopaminergic-like (DALNs, ~26% TH+/DAT+ flow cytometry) and cholinergic-like neurons (ChLNs, ~46% ChAT+/VAChT flow cytometry) which responded to dopamine- and acetylcholine- triggered neuronal Ca 2+ inward stimuli when cultured with the NeuroForsk and the Cholinergic-N-Run medium , respectively in a timely fashion (i.e., 4-7 days). Here, we also report a direct transdifferentiation method to induce MenSCs into functional astrocyte-like cells (ALCs) by incubation of MenSCs in commercial Gibco® Astrocyte Medium in 7-days. The MSCs-derived ALCs (~59% GFAP+/S100b+) were found to respond to glutamate-induced Ca 2+ inward stimuli. Altogether these results show that MenSCs are a reliable source to obtain functional neurogenic cells to further investigate the neurobiology of neurologic disorders.

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OCT-1 Protein Expression Detected by Flow Cytometry Provided Valuable Information in CML Patients Treated with Imatinib MesylateÃ,Â FHigher hOCT-1 Expression Predict Better Outcome.

Blood ◽

10.1182/blood.v114.22.1117.1117 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1117-1117

Author(s):

Huanling Zhu ◽

Qiurong Zhang ◽

Nenggang Jiang ◽

Ting Liu

Keyword(s):

Flow Cytometry ◽

Protein Expression ◽

Conflicts Of Interest ◽

Organic Cation Transporter ◽

Negative Control ◽

Expression Level ◽

Molecular Response ◽

Fluorescent Intensity ◽

Healthy Donors ◽

Significant Difference

Abstract Abstract 1117 Poster Board I-139 Objective The human organic cation transporter-1(hOCT-1) is the major active influx protein responsible for the transport of imatinib into cells. The functional activity of the hOCT-1 protein using 14-C detected by others demonstrated a link between CML molecular response and hOCT-1 activity. However, 14-C labeled detection is not convenient in routine clinical practice. Hence, we use flow cytometry to detect hOCT-1 protein expression level in CML patient and try to find some relation between hOCT-1 expression and imatinib response. Subjects and methods In this study, 64 CML CP patients and 31 healthy donors were enrolled. Totally, there are 78 patient' peripheral blood (PB) or bone marrow (BM) samples and 31 donor PB samples were measured. The hOCT-1 protein expression levels were detected by indirect immunofluorescent flow cytometry. The hOCT-1 levels were expressed as mean fluorescent intensity (MFI). In avoided to systematic error, lymphocytes which had little hOCT-1 expression were used as internal negative control. Results ‡@ Assessing PB hOCT-1 expressing in patients with donors, hOCT-1 level is higher in healthy donors than in CML patient (mean±standard deviation 9.11±6.04,5.60±3.74,P=0.005). ‡AThe hOCT-1 level was compared with molecular response in patients. Of 39 patients achieved optimal molecular response, the hOCT-1 level was 6.49±3.83, versus 3.86±2.91 in 20 patients with non-optimal response(P=0.009). Comparing 39 optimal responders with 16 sub-optimal responders, hOCT-1 level were 6.49±3.83, versus 3.98±1.23(P=0.025. ‡B Assessing CML stages with hOCT-1 expression, there is no significant difference in chronic stage and advanced stage(5.93±3.87, 3.49±1.64, P=0.085). Conclusions hOCT-1 expression level measured by flow cytometry is very convenient and clinically available. The hOCT-1 expression level can be an important predictor in CML patients treated with imatinib mesylate. Disclosures No relevant conflicts of interest to declare.

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The Sesquiterpene Oil α-Bisabolol Induces Apoptosis of B-Chronic Lymphocytic Leukemia Primary Cells

Blood ◽

10.1182/blood.v118.21.1319.1319 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1319-1319

Author(s):

Massimiliano Bonifacio ◽

Antonella Rigo ◽

Angela Bonalumi ◽

Emanuele Guardalben ◽

Ilaria Nichele ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Lipid Rafts ◽

Conflicts Of Interest ◽

Membrane Integrity ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Primary Cells

Abstract Abstract 1319 We have recently demonstrated that the sesquiterpene oil α-bisabolol is cytotoxic against primary acute leukemia cells ex vivo and in chronic myeloid leukemia cell lines. It enters cells via lipid rafts and activates the mitochondrial-dependent intrinsic pathway of apoptosis, exerting a preferential toxicity against malignant vs normal cells probably due to their higher content in lipid rafts. Here we investigated the in vitro activity of α-bisabolol in primary cells from patients with B-Chronic Lymphocytic Leukemia (B-CLL). Twenty-six patients with newly diagnosed B-CLL gave their informed consent to the study. Cells were collected before any treatment, purified and cultured for 24 hours with serial dilutions of α-bisabolol. Citotoxicity was quantified in flow cytometry by the BD Trucount™ technology to allow comparison between neoplastic and normal residual lymphocytes. B-CLL cells (IC50 42±15 μM) were significantly more sensitive towards α-bisabolol than normal B- (IC50 82±34 μM, p=.005) and T-cells (IC50 120±35 μM, p<.001). Citotoxicity was similar between the IgVH mutated (n=11) and the IgVH unmutated samples (n=7), as well as between the Binet stage A (n=20) and B-C (n=6) patients. To investigate the mechanisms of α-bisabolol-induced toxicity we treated B-CLL cells with 40 μM α-bisabolol for up to 3 hours. We observed a time-dependent increase in fluorescence of cells treated with the membrane-impermeant nucleic acid stain TO-PRO-3, already detactable after 30 minutes. When cells were loaded with the Ca2+ indicator Fluo-4 AM, an increase of Ca2+ influx was revealed already after 15 minutes. These early events indicate that α-bisabolol induces the loss of cellular membrane integrity, so triggering the apoptotic cascade. Then we assessed the mitochondrial transmembrane potential (ΔΨm) with the fluorochrome JC-1 to confirm that a mitochondrial damage is a concurrent mechanism in the apoptotic process induced by α-bisabolol. By flow cytometry we demonstrated that, after 3-hour incubation with 40 μM α-bisabolol, ΔΨm dissipation was already detectable in leukemic cells, while T-lymphocytes, evaluated as internal control in the same samples, stayed vital. To investigate the mitochondrial target of α-bisabolol we examined the function of the mitochondrial permeability transition pore (mPTP). After 5-hour incubation with 40 μM α-bisabolol we loaded cells with the calcein AM dye and added CoCl2 to distinguish between intact and damaged mitochondria, confirming that the function of mPTP was compromised in B-CLL cells but not in normal controls. Finally, to determine whether α-bisabolol affects the oxydative state of treated cells, we evaluated the intracellular concentration of reactive oxygen species (ROS) by measuring the fluorescent signal of CM-H2DCFDA loaded cells. When B-CLL cells were exposed to 40 μM a-bisabolol for 3 hours, they exhibited a clear fluorescence increase, indicating the striking generation of ROS: this was completely abrogated by the addition of N-acetylcysteine, a scavenger of intracellular ROS. Clues about the molecular mechanistics of α-bisabolol have also emerged from in vitro models based on treating cells previously transfected with BH3-only molecules. In this setting, α-bisabolol exposed cells seem to undergo detrimental, non-selective autophagy-like phenomena. Our data indicate that α-bisabolol exerts a level of cytotoxicity against B-CLL cells at concentrations that only partially affect normal B- and T-cells. Moreover, a brief exposure (3–5 hours) to α-bisabolol is sufficient to elicit multiple pro-apoptotic signals independently of the patients' mutational status. Disclosures: No relevant conflicts of interest to declare.

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Rat spermatogenesis in vitro traced by quantitative flow cytometry.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.8.3734416 ◽

1986 ◽

Vol 34 (8) ◽

pp. 1029-1035 ◽

Cited By ~ 19

Author(s):

J Toppari ◽

P Mali ◽

E Eerola

Keyword(s):

Flow Cytometry ◽

Morphological Differentiation ◽

Culture Conditions ◽

Seminiferous Tubules ◽

Dna Flow Cytometry ◽

Cell Numbers ◽

Pachytene Spermatocytes ◽

Quantitation Method ◽

Rat Spermatogenesis

In vitro differentiation of germ cells in rat seminiferous tubule segments at stages II-III of the epithelial cycle was studied. DNA flow cytometry was used for quantitation of absolute cell numbers from the cultured tubule segments that were compared to freshly isolated stages of the cycle, as identified by transillumination stereomicroscopy of the seminiferous tubules and phase-contrast microscopy of live cell squashes. Spermatogonia and spermatocytes from stages II-III showed normal morphological differentiation during 7 days in vitro. Round spermatids differentiated to Step 7 of spermiogenesis but Step 16 spermatids failed to develop. Acid phosphatase activity in the spermatogenic cells changed normally during the culture. As compared with freshly isolated control tubule segments, 35% of round spermatids and 42% of pachytene spermatocytes were present in culture after 7 days. The cell numbers recovered from defined stages by DNA flow cytometry were close to those found in morphometric studies. Flow cytometry is an efficient quantitation method for cells liberated from seminiferous epithelium. Spermatogonia, spermatocytes, and early spermatids are able to differentiate in vitro, but spermatids approaching the elongation (acrosome) phase, and particularly the maturation phase, fail to differentiate under present culture conditions.

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Leukemia Cells Remodel Adipocyte Niches and Their Progenitor Functions to Generate Leukemia Favoring Niche

Blood ◽

10.1182/blood-2018-99-115689 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1294-1294

Author(s):

Bijender Kumar ◽

Marvin Orellana ◽

Jamison Brooks ◽

Srideshikan Sargur Madabushi ◽

Liliana E Parra ◽

...

Keyword(s):

Flow Cytometry ◽

Conflicts Of Interest ◽

Leukemia Cells ◽

Lymphoid Leukemia ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Maturation Defect ◽

Induced Apoptosis

Abstract Increasing evidence suggests that the cancer cells take shelter in different osteoblastic and adipocytic niches, where they hide from chemotherapy and continue to survive. As yet, how leukemia cells alter the bone marrow (BM) adipocytic niches to facilitate their expansion and assist them in evading chemotherapy is unclear. We have previously shown that the acute myeloid leukemia (AML) cells directly or through their exosomes, reprogram BM osteoblastic niche which facilitates their expansion and suppress normal hematopoiesis(Kumar B et al, Leukemia 2018,32(3):575-587). In this study , we provide further evidences that AML and Acute lymphoid leukemia (ALL) transformed the BM adipocytic niche to facilitate their expansion and suppress normal hematopoiesis. Using MLL-AF9 (AML) knock-in mouse, MLL-AF9 or BCR-ABL(p190, ALL) HSC transduction transplantation leukemia mice models, we performed flow cytometry analysis to show that the leukemia cells stimulate expansion of BM derived CD45-Ter119-CD31-CD166-Sca1+CD140a+(PaS)MSCs population compared to normal mice (p=0.04, p=0.001 and p=0.002 respectively).Further, the BM osteoblasts specific Osteocalcin mRNA expression in sorted stroma cells and flow cytometry based osteoblasts population (CD45-Ter119-CD31- CD166+Sca1-) numbers were also significantly reduced in AML (p=0.04) and ALL (p=0.02) mice models suggesting bone loss with the leukemia development. Similar to osteoblasts loss, mature adipocytes (Perilipin, PPARg mRNA) were also significantly reduced in the ALL/AML mice compared to control. The triglyceride content and white adipose tissue(WAT) mass was diminished in leukemic mice , suggesting leukemia may have utilized adipocyte for survival. Adipocyte loss in the leukemia mice was accompanied by long term hematopoietic stem cells(LT-HSC ) and erythroid megakaryocyte progenitor (MEP) populations reduction in the leukemic mice (p=0.01 and p=0.02 respectively). To dissect the mechanism of adipocytes reduction is either due to adipocyte loss or adipocytes maturation defect in leukemic mice, we analyzed different stromal progenitors in normal and leukemic mice and identified that the leukemia cells stimulate the growth of BM derived adipocytic committed progenitors (CD45-Ter119-CD31-CD166-Sca1+CD140a+CD29+CD24-) and blocked the chondrocyte/osteoblastic/adipocytic multipotent progenitors (CD45-Ter119-CD31- CD166-Sca1+CD140a+CD29+CD24+) population (p=0.02,p=0.01 respectively).Despite the increase in number of MSCs and adipocytic progenitors, the in-vitro adipocytic differentiation potential of sorted adipocyte committed progenitors was severely compromised in ALL and AML compared to control. The WAT western blot analysis showed significantly increased expression of ATGL and pHSL(Ser-563) expression involved in triglyceride lipolysis in the leukemic mice .The ALL leukemic adipocytic stroma had increased expression of IL-1β and IL-6 cytokine levels compared to normal stroma and provided more survival advantage to leukemia cells in in-vitro co-culture experiments in nutrient deprived conditions and during chemo-radiotherapy treatment. Further, the ATGL and HSL pharmacological inhibitors rescued leukemia induced lipolysis, reduced leukemia proliferation and increased chemotherapy induced apoptosis in leukemia cells. Overall, this data strongly suggests the notion of progressive decline in functional LT-HSCs & normal hematopoiesis, adipocytes and osteoblasts numbers with leukemia progression due to activation of lipolytic enzymes resulting in increased availability of fatty acids for leukemia expansion and is a common feature in both lymphoid and myeloid leukemias. Disclosures No relevant conflicts of interest to declare.

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Identification of Pre-Gmps and Pre-Meps: Two Novel Types of Committed Progenitor Cells in Bone Marrow

Blood ◽

10.1182/blood-2021-153800 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3266-3266

Author(s):

Ryan Mack ◽

Lei Zhang ◽

Kanak Joshi ◽

Shanhui Liu ◽

Mark Sellin ◽

...

Keyword(s):

Conflicts Of Interest ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Effector Cells ◽

Functional Studies ◽

Lineage Differentiation ◽

Reliable Marker ◽

Relationship Of

Abstract Elucidating the stepwise differentiation processes that leads from multipotent hematopoietic stem cells to mature effector cells is critical for understanding both normal and neoplastic hematopoiesis. Early studies suggested that common myeloid progenitors (CMPs) are oligo-lineage hematopoietic progenitors that produce all lineages of myeloid cells, including granulocytes, monocytes, erythrocytes and megakaryocytes. CMPs do so by first giving rise to megakaryocyte-erythroid progenitors (MEPs) and granulocyte-monocyte progenitors (GMPs), two types of bi-lineage progenitors. However, this concept was challenged by several recent studies where single cell techniques demonstrated that CMPs, GMPs and MEPs are highly heterogenic. The existence of lineage-restricted subsets within the CMP population leads to questions about whether erythroid and megakaryocytic lineage commitment is actually initiated at the multipotent progenitor or CMP stage. During the past 15 years, several lineage-restricted subsets of progenitors have been separated out from CMP population, including monocyte-dendritic progenitors, megakaryocyte progenitors, and erythroid progenitors based on expression of CD115/Flt3, CD41/CD150, and CD105/CD150, respectively. However, the remaining CMP population is still highly heterogenic. Thus, further separation of functional subsets within the CMP compartment is required. By screening cell surface markers that can further separate CMPs, we have identified CD27 as a reliable marker to separate all megakaryocyte/erythrocyte-committed progenitors from granulocyte/monocyte-committed progenitors. In addition, we found that CD62L is only expressed on granulocyte/monocyte-committed progenitors. CD27 and CD62L co-staining can separate CMP into CD27 +CD62L +, CD27 +CD62L - and CD27 -CD62L - subsets. Biology and morphology study showed that CD27 +CD62L - cells are closely associated with GMPs, whereas CD27 -CD62L - cells are closely associated with MEPs. In vitro culture and in vivo transplantation functional studies demonstrated that 1) CD27 +CD62L + cells are pre-GMPs that give rise to FcGRII/III + GMPs and only produce granulocytes and monocytes; 2) CD27 -CD62L - cells are pre-MEPs that give rise to MEPs and primarily produce erythrocytes and megakaryocytes with minimal contribution to granulocytes and monocytes; 3) CD27 +CD62L - subset enriches cells with genuine CMP potential capable of producing GMPs, MEPs, and subsequent progeny. Taken together, we have identified two novel populations of committed progenitors that serve as intermediates between CMP-GMP and CMP-MEP commitment pathways. Identification of pre-GMPs and pre-MEPs fills in the gap between CMPs-GMPs and CMPs-MEPs, supporting the hierarchal relationship of myeloid lineage differentiation. Disclosures No relevant conflicts of interest to declare.

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