Jolene Bradford

Development and Characterization of a Clinically Compliant Xeno-Free Culture Medium in Good Manufacturing Practice for Human Multipotent Mesenchymal Stem Cells

Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohns disease, multiple sclerosis, graft‐versus‐host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum‐free media that contain serum albumin, insulin, and transferrin. The ill‐defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno‐free culture media to meet the regulatory and long‐term safety requirements for cell‐based therapies. We developed and tested a serum‐free and xeno‐free culture medium (SFM‐XF) using human bone marrow‐ and adipose‐derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM‐XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum‐containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow‐derived MSCs expanded in SFM‐XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM‐XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM‐XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum‐containing media for the production of large scale, functionally competent MSCs for future clinical applications.

Dual‐Pulse Labeling Using 5‐Ethynyl‐2′‐Deoxyuridine (EdU) and 5‐Bromo‐2′‐Deoxyuridine (BrdU) in Flow Cytometry

Changes in DNA replication during S‐phase can give insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation utilizes the incorporation of a thymidine analog during DNA synthesis. Incorporation of multiple analogs at different time points can further define cell cycle kinetics. Traditionally, the dual‐pulse method has been done by combining 5‐bromo‐2′‐deoxyuridine (BrdU) with iododeoxyuridine or chlorodeoxyuridine, with detection using multiple cross‐reacting BrdU antibodies. This unit presents a dual‐pulse method using the thymidine analog 5‐ethyl‐2′‐deoxyuridine (EdU), detected by click chemistry, combined with BrdU labeling and detection. No cross reactivity with incorporated EdU is observed using the BrdU antibody clone MoBU‐1. EdU detection using click chemistry does not cross‐react with incorporated BrdU. Cells are first pulsed with EdU, and then pulsed with BrdU; sequential pulses of EdU, followed by BrdU, are done without removing or washing out EdU. Curr. Protoc. Cytom. 55:7.38.1‐7.38.15.

Panel Development for Multicolor Flow-Cytometry Testing of Proliferation and Immunophenotype in hMSCs

Adult human mesenchymal stem cells (hMSC) are rare fibroblast-like cells capable of differentiation into a variety of cell tissues which include bone, cartilage, muscle, ligament, tendon, and adipose. Normal adult bone marrow and adipose tissue are the most common sources of these cells. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSC for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; and specific surface antigen expression. Direct measurement of proliferation combined with simultaneous detection of the ISCT-consensus immunophenotypic profile provides data that is used to determine the differentiation status and health of the cells. Flow cytometry provides a powerful technology that is routinely used to simultaneously and rapidly measure multiple parameters in a single sample. This chapter describes a flow cytometric panel for the simultaneous detection of immunophenotypic profile, proliferative capacity, and DNA content measurement in hMSC. Because a relatively small number of cells are needed with this approach, measurements can be made with minimal impact on expansion potential. The ability to assess antigen expression and proliferative status enables the investigator to make informed decisions on expansion and harvesting.

Is alkaline phosphatase the smoking gun for highly refractory primitive leukemic cells

With the aim to detect candidate malignant primitive progenitor populations, we modified an original alkaline phosphatase (ALP) stem cell detection method based on the identification of alkaline phosphatase fluorescent cells in combination with flow cytometry immunophenotyping. Over a period of one year, we have been using this technique to study its activity in patients with leukemia and lymphoma, showing that changes in the alkaline phosphatase levels can be used to detect rare populations of highly refractory malignant cells. By screening different blood cancers, we have observed that this activity is not always restricted to CD34+ leukemic cells, and can be overexpressed in CD34 negative leukemia. We have verified that this method gives accurate and reproducible measurements and our preliminary results suggest that CD34+/ALPhigh cells appear to sustain leukemogenesis over time.

Acoustophoretic Orientation of Red Blood Cells for Diagnosis of Red Cell Health and Pathology

Distortions of the normal bi-concave disc shape for red blood cells (RBCs) appear in a number of pathologies resulting from defects in cell membrane skeletal architecture, erythrocyte ageing, and mechanical damage. We present here the potential of acoustic cytometry for developing new approaches to light-scattering based evaluation of red blood cell disorders and of the effects of storage and ageing on changes or damage to RBCs membranes. These approaches could be used to immediately evaluate the quality of erythrocytes prior to blood donation and following transfusion. They could also be applied to studying RBC health in diseases and other pathologies, such as artificial heart valve hemolysis, thermal damage or osmotic fragility. Abnormal distributions of erythrocytes can typically be detected after just 30 to 45 seconds of acquisition time using 1–2 µL starting blood volumes.

Abstract 4724: No-lyse no-wash immunophenotyping.

Immunophenotyping whole blood is a primary application in the study of blood cell populations and their functions. Red blood cells have traditionally been removed during sample preparation by lysis methods or density-gradient selection to enable the study of nucleated cell populations. This processing of whole blood has its draw backs due to the potential loss of cells of interest thru inadvertent lysis and unintentional selection of fragile and rare cells including blast cells, cancer cells, and cancer stem cells. The application of a No-Lyse No-Wash protocol in human whole blood samples benefit from a streamlined protocol that reduces cell loss. Testing of mouse whole blood presents additional challenges for cancer researchers due to the limited sample volume available (≤100 μL/day/animal), particularly in longitudinal studies. Specifically, these small volumes limit the ability to perform immunophenotyping experiments with required controls. This work describes a No-lyse No-wash method which takes advantage of acoustic focusing technology. Immunophenotyping panels are shown for both human and mouse whole blood. A total of 10μL of whole blood is stained in a 50 μL total volume and then diluted 400-fold in PBS. A fluorescence threshold is used to distinguish the white blood cell populations from the more abundant red blood cell population. In the mouse immunophenotyping example, CD45 is used to threshold on white blood cells. For the human immunophenotyping example, a cell permeable nucleic acid stain is used to threshold on all nucleated cells. At this dilution, the coincidence of the target population with red blood cells is reduced sufficiently so scatter signals are reliable for population identification. The significantly higher sample collection rates available with acoustic cytometry allows for the acquisition of high quality data utilizing a No-lyse No-wash protocol in a timely manner. Difficult-to-collect and precious samples like mouse blood can be stained and diluted without washing or performing RBC removal procedures. Loss of cells due to sample preparation is essentially eliminated, and ability to detect the cells of interest is enabled. Citation Format: Jolene A. Bradford, Rick Kerndt. No-lyse no-wash immunophenotyping. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4724. doi:10.1158/1538-7445.AM2013-4724

Abstract 2380: Detecting human circulating endothelial cells using the acoustic focusing cytometer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Circulating endothelial cells (CECs) are mature cells shed from blood vessel walls during the natural process of endothelial cell turnover. Elevated levels of CECs have been reported in a host of pathological conditions including cardiovascular disorders, infectious diseases, immune disorders, post-transplantation analysis, and cancer. In cancer research, CECs have been suggested as a noninvasive biomarker for angiogenic activity, providing insight into tumor regrowth, resistance to chemotherapy, early recurrence and metastasis formation during or after chemotherapy. In healthy individuals, CECs are reported to be present in very low numbers: 0.01%-0.0001% of all peripheral blood mononuclear cells. Flow cytometry offers the necessary collection and analysis capabilities for detection of CECs, but is subject to numerous technical challenges. In comparison to traditional hydrodynamic-focusing cytometers, the Attune® Acoustic Focusing Cytometer, with its fast acquisition times and increased precision, overcomes the technological hurdles involved in analyzing CECs. Here we present a method for detecting human circulating endothelial cells using the Attune® Acoustic Focusing Cytometer. The method includes a number of conventional ways to improve rare event detection: a blocking step, a viability stain (SYTOX® AADvanced™ Dead Cell Stain), and the use of a dump channel to eliminate unwanted cells and decrease background fluorescence. The challenge of collecting a large enough number of events in a reasonable amount of time is met by using a collection rate of 1000 µL/min with the Attune® cytometer. This setting enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes; the acquisition time using a traditional hydrodynamic-focusing cytometer would be 10-12 times longer, close to 6 hours. Furthermore, this method delivers additional time savings by eliminating wash steps to avoid sample loss and employing a simpler sample preparation method; details and specific panels designed for both the blue/violet and blue/red Attune® cytometer configurations are outlined. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2380. doi:1538-7445.AM2012-2380

Abstract 4896: Acoustic cytometry for rare event detection of PNH cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Rare event analysis is an area of broad interest in pathology. In hematopathology, applications include detection of residual leukemic cells in peripheral blood, bone marrow and other fluids, and levels of circulating CD34+ stem cells in blood. An additional area of recent interest is detection of small populations of neutrophils in the blood which lack surface expression of certain markers due to absence of a glycophosphatidylinositol (GPI)-anchor protein. In classic paroxysmal nocturnal hemoglobinuria (PNH), a clone of hematopoeitic stem cells arises which expands in most cases to >80% of marrow cells. Small PNH-like neutrophil populations compromising 1.0 – 0.01% of neutrophils have been detected in the blood of up to 15% of patients who have myelodysplasia (MDS) and 24% of patients with aplastic anemia. These patients do not progress to classic PNH, and do not have the clinical sequelae of hemolytic anemia or thrombosis of PNH. The finding of these minor PNH-like neutrophil populations is of clinical interest because the finding correlates with increased sensitivity to immunosuppressant therapy. There are now preliminary consensus guidelines recommending routine PNH testing on all aplastic anemia and MDS patients who are candidates for therapy. Flow cytometry is the method of choice for detection of PNH cells, made possible with technological improvements in instrumentation combined with fluorescent reagents to study GPI-linked antigens on cells. One of the practical issues complicating rare event detection and analysis is the need to acquire a very large number of events in order to obtain statistically meaningful data, which can require excessive acquisition time. The current detection limit with conventional flow cytometry using hydrodynamic focusing for rare event detection is in the range of 0.1-0.01% of nucleated cells, accumulating 1 million total events over a 60 minute interval per sample. A recent advance in cytometry instrumentation utilizes acoustic focusing, in which acoustic energy is used to precisely align cells for more sensitive detection, and sample acquisition time can be reduced by 10-fold or more. The detection limit with acoustic focusing cytometry is in the range of 0.1-0.01% of nucleated cells, accumulating 1 million total events over a 5 minute interval per sample. We present an approach for the detection of PNH-like neutrophils using acoustic cytometry. Utilizing a panel of fluorescent reagents including CD45, CD15, CD16, CD24 and fluorescent aerolysin (FLAER) surface markers, a gating strategy is employed for high-sensitivity analysis of neutrophils using lineage markers. FLAER binds specifically to GPI anchor-deficient cells and is the most useful reagent for detecting PNH neutrohpils. Results of both normal and abnormal patient samples are shown. Acoustic cytometry provides the ability to detect PNH neutrophils by acquiring statistically significant cells for rare event detection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4896. doi:10.1158/1538-7445.AM2011-4896

Abstract 1943: A comparison of three techniques to induce efficient ex vivo T-cell expansion

The current study was undertaken to evaluate three common methods of inducing proliferation in a T lymphocyte population. In the first method cells are treated with purified monoclonal mouse anti-human CD3 antibody followed by addition of interleukin-2. The second approach involves treatment with the mitogen Concanavalin A. The third technique makes use of monoclonal mouse antibodies against human CD3 and CD28 immobilized on the surface of superparamagnetic polystyrene microspheres. Human peripheral blood mononuclear cells were isolated from fresh whole blood using a centrifuge density gradient. Cells were stained with CellTrace™ Violet, a novel cell proliferation tracing dye with a mode of action similar to carboxyfluorescein diacetate succinimidyl ester (CFSE), and resuspended in OpTmizer T-Cell Expansion Buffer (GIBCO) containing L-glutamine, penicillin and streptomycin. Stained cells were treated with one of the three stimulation techniques and incubated at 37°C and 5% CO 2 for 7 days. A cell impermeant nucleic acid dye was used to evaluate the effect of each treatment on cell viability. The specific subpopulations induced to proliferate with each treatment were characterized in multicolor flow cytometry using monoclonal antibody conjugates against human CD3, CD4, and CD8. Cells treated with CD3/IL-2 or Concanavalin A showed a moderate, heterogeneous level of stimulation, with some cells undergoing 9 cell divisions, others remaining senescent, and approximately equal amounts remaining in generations 1 through 8. Microsphere-bound antibodies provided a much stronger, more uniform stimulus as evidenced by the very high proliferative index of cells. Concanavalin A treatment was mildly cytotoxic, while viability of cells treated with free or bound antibodies was similar to unstimulated cells. The strong, homogeneous stimulus provided by superparamagnetic polystyrene expansion beads is useful when massive numerical expansion of T lymphocytes is desired for subsequent research or adoptive transfer in vivo, but does not appear to be as useful when a population containing several discrete generations of cells is desired. The more moderate stimuli resulting from treatment with CD3/IL-2 or Concanavalin A result in a less efficient numerical expansion, but provide an excellent tool to evaluate the phenotype and pedigree of cells as they proliferate through several generations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1943.

Abstract 3976: A novel violet-laser excitable ratiometric probe for the detection of membrane asymmetry breakdown during apoptosis

Apoptosis is carefully regulated and essential part of normal tissue development and homeostasis. Regulatory changes in the apoptotic pathway have been implicated in many diseases; most notable are many types of cancers and autoimmune disorders. Normal cells exhibit a remarkable asymmetry in lipid distribution between the outer and inner cell membranes characterized by phosphatidyl-serine (PS) and phosphatidylethanolamine (PE) normally located on the inner leaflet of the cell membrane. During apoptosis translocation of PE and PS to the external cellular environment facilitates recognition and elimination of these cells by macrophages. The Violet Ratiometric Membrane Asymmetry Probe, 4′-N, N-diethylamino-6-(N, N, N-dodecyl-methylamino-sulfopropyl)-methyl-3-hydroxyflavone (F2N12S), is a novel violet excitable dye for the detection of membrane asymmetry changes during apoptosis. The dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction resulting in a dual fluorescence with two emission bands corresponding to 530 nm and 585 nm, producing a two-color ratiometric response to variations in surface charge. This ratiometric probe is a self-calibrating absolute parameter of apoptotic transformation, which is independent of probe concentration, cell size, and instrument variation. The two-color ratiometric response of the F2N12S reagent will detect apoptotic cells in samples that vary in cell concentration by as much as 100 fold. The Violet Ratiometric Membrane Asymmetry Probe rapidly targets the plasma membrane requiring only a 5 minute room temperature incubation without additional buffers or wash steps reducing the chance of cell death that can occur from prolonged sample processing. The Violet Ratiometric Membrane Asymmetry Probe will work in both suspension and adherent cell lines and its efficacy is confirmed by caspase and mitochondrial apoptosis markers. Annexin V staining produced a larger population of apoptotic cells during early time points in both suspension and adherent cell lines that do not correlate with caspase and mitochondrial apoptosis markers. The violet 405 nm excitation of F2N12S expands the capability of multi-parameter apoptosis assays to study the apoptotic process while reducing or eliminating the need to create complex compensation controls by taking advantage of the time and spectral separation a violet-excitable reagent provides. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3976.