Cell-Surface Marker Signatures for the Isolation of Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations

B cells in autoimmune (NZB × NZW)F1 mice show altered IgG isotype switching upon T cell-dependent antigenic stimulation in vitro

Humoral immune responses of (NZB × NZW)F1 (BWF1) autoimmune mice to T cell-dependent antigens often exhibit a predominance of IgG2 antibodies, while normal mice produce IgG1 antibodies. In order to determine whether this results from differences in properties of the B cells or the T cells involved, the responses of both primary and secondary BWF1 B cells to the antigen DNP-hemocyanin (Hy) were measured in limiting dilution splenic fragment cultures in the presence of normal T cell help. Furthermore, the capacity of Hy-primed lymph node T cells from BWF1 mice to provide help to BALB c nu nu B cells was determined in modified splenic fragment cultures. These experiments indicated that (a) stimulation of primary BWF1 B cells with DNP-Hy and normal T cell help failed to yield significant numbers of clones which produced any of the IgG isotypes; (b) antigenic stimulation of BWF1 secondary B cell clones also demonstrated a paucity of IgG1, but elevated production of IgG2 isotypes; and (c) Hy-primed BWF1 lymph node T cells were comparable to those derived from BALB c mice in their capacity to provide both help for nu nu B cell responses and modulation of IgG isotype switching. BWF1 B cells apparently differ from normal murine B cells in their capacity to produce IgG antibodies upon T cell-dependent antigenic stimulation

The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.

BACKGROUND: Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. CONCLUSIONS/SIGNIFICANCE: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research

Multiplex Flow Cytometry Barcoding and Antibody Arrays Identify Surface Antigen Profiles of Primary and Metastatic Colon Cancer Cell Lines

<div><p>Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.</p> </div

CD10 expression in SW480 versus SW620.

<p>Histogram plots from antibody array for the CD10 antigen in SW480 (<b>A</b>) and SW620 (<b>B</b>). Red indicates isotype control while the blue line is staining for CD10. The number in the top left is the cell positivity. There is a clear shift from a small shoulder population in SW480 to complete binding in SW620 cells. <b>C</b>) Immunoblotting for CD10 confirms the strong change in CD10 expression.</p

Expression of surface stem cell markers.

<p>Expression of putative surface cancer stem cell markers (% of live cells) in colon cancer cell lines as detected by multicolor flow cytometry.</p

Diagram of experimental methods used for multiplexed barcoded antibody array.

<p>The three cell lines were labeled with or without intracellular dye prior to admixing the cells into a single pool. The cells were then aliquoted into each well for antibody labeling. The contents of each well were then processed on a flow cytometer. The identity of each cell line was determined based on fluorescence intensity. The appropriate gates were drawn allowing for simultaneous analysis for each antibody. Histograms for mouse IgM isotype control are shown.</p

Validation of integrin α6 expression in colon cancer by immunohistochemistry.

<p><b>A</b>) H&E (top left) and integrin α6 IHC (top right) from clinical colon cancer specimens at low magnification. Areas of normal mucosa (N) and adjacent primary colon cancer (P) are indicated. Lower panels provide higher magnification fields of integrin α6 in normal (left) and tumor (right). <b>B</b>) Representative examples of liver and lymph node metastases. The regions of colon cancer metastases (M) are visible by H&E (left) and corresponding staining with integrin α6 (right). An area of normal liver (L) is indicated. All lymph node samples contained a high degree of fibrosis around the lesion that displaced normal lymphoid tissue from the field of view. Scale bar: 50 µm.</p

Broadly expressed tumor antigens.

<p>Antibody array results showing surface antigens that were expressed on at least 50% of cells in all three colon cancer cell lines analyzed. Arranged in alphanumeric order.</p>*<p>, Antigens common to all nucleated human cells.</p><p>Abbreviations: lymphocyte function-associated antigen 3, LFA-3; membrane inhibitor of reactive lysis, MIRL; transferrin receptor protein 1, TFRC; large neutral amino acid transporter 1; LAT1; melanoma cell adhesion molecule, MCAM; basigin, BSG; L1 cell adhesion molecule, L1CAM; common leukocyte antigen, CLA.</p