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CD34+Thy-1+Lin− cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34+Thy-1+Lin− cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34+Thy-1+Lin− cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34hiPKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34+Thy-1+Lin− cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34hi PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34hiPKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34+Thy-1+Lin− cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.

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Mass cytometry is an emerging technology capable of 40 or more correlated measurements on a single cell. The complexity and volume of data generated by this platform have accelerated the creation of novel methods for high-dimensional data analysis and visualization. A key step in any high-level data analysis is the removal of unwanted events, a process often referred to as data cleanup. Data cleanup as applied to mass cytometry typically focuses on elimination of dead cells, debris, normalization beads, true aggregates, and coincident ion clouds from raw data. We describe a probability state modeling (PSM) method that automatically identifies and removes these elements, resulting in FCS files that contain mostly live and intact events. This approach not only leverages QC measurements such as DNA, live/dead, and event length but also four additional pulse-processing parameters that are available on Fluidigm Helios™ and CyTOF ® (Fluidigm, Markham, Canada) 2 instruments with software versions of 6.3 or higher. These extra Gaussian-derived parameters are valuable for detecting well-formed pulses and eliminating coincident positive ion clouds. The automated nature of this new routine avoids the subjectivity of other gating methods and results in unbiased elimination of unwanted events. CYTOF instruments were invented as an alternative technology to flow cytometry (1,2). Instead of fluorescent molecules, heavy metals are conjugated to antibodies, which bind to specific epitopes on cells (3). Metal-labeled cells are detected and quantified by inductively coupled plasma mass spectrometry (ICP-MS) with time-of-flight detection (4). Because this technology avoids spectral overlap of fluorescent dyes and isotopic metal contamination is well less than 5%, the number of correlated measurements is mainly limited by the number of stable isotopes of rare earth metals, which realistically could soon reach 100 or more (5).Whether a cytometer is fluorescence-based or metal-based, there are always undesired events that need to be eliminated prior to analysis. Typical flow cytometers have internal circuitry or logic that ignores signal-derived pulses that are partially formed or abnormally long. Flow cytometry often employs a forward by 90 light-scatter gate to eliminate debris and aggregates. Also, pulse processing features such as peak height, width, and area can be leveraged to reduce the number of aggregates.Since mass cytometry atomizes particles into clouds of positively charged ions, its pulse processing capabilities are mainly targeted at detecting and eliminating coincident ion clouds or poorly formed pulses. Mass cytometry also has DNA intercalators (1) that can eliminate debris and some true aggregates. Both technologies 1 Verity Software House, Topsham, Maine

High‐dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30‐marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™. In this study, seven sites received whole blood and six sites received PBMC samples from single donors over a 2‐week interval. Each site labeled replicate samples and acquired data on Helios™ instruments using an assay‐specific acquisition template. All acquired sample files were then automatically analyzed by Maxpar Pathsetter™ software. A cleanup step eliminated debris, dead cells, aggregates, and normalization beads. The second step automatically enumerated 37 immune cell populations and performed label intensity assessments on all 30 markers. The inter‐site reproducibility of the 37 quantified cell populations had consistent population frequencies, with an average %CV of 14.4% for whole blood and 17.7% for PBMC. The dry reagent coupled with automated data analysis is not only convenient but also provides a high degree of reproducibility within and among multiple test sites resulting in a comprehensive yet practical solution for deep immune phenotyping.

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34+ cells lacking lineage differentiation markers (CD34+Lin−) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34+ cell population retained the Lin− phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34+Lin−NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34+Lin−NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% ± 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.