Name: isolation of precursor b-cell subsets from umbilical cord blood
Uploaded: 2013-04-16T13:00:00
Duration: 14 min 6 s
Description: Watch this Scientific Journal Video about Isolation of Precursor B-cell Subsets from Umbilical Cord Blood at JoVE.com

This work was supported by the National Institutes of Health (NCI R00 CA132784) to K.T.

1. Isolation of Mononuclear Cells from Umbilical Cord Blood
<ol>
 <li>Prepare EDTA-PBS buffer- add 5 ml bovine serum albumin (BSA) stock solution to 95 ml rinsing buffer (1:20 dilution). Degas the buffer and keep the buffer on ice. IMPORTANT: Failure to degas the buffer may result in less than optimal results when isolating CD19+ B-cells because bubbles may block the isolation column.</li>
 <li>Prepare 50 ml conical tubes for density gradient centrifugation. Determine the number of tubes required for processing the cord blood (1 tube can process 8 ml blood) and add 15 ml of Ficoll-Paque PLUS to each tube.</li>
 <li>Dilute 8 ml of cord blood with 24 ml DPBS (1X) and carefully layer the diluted cord blood mixture on top of the Ficoll-Paque PLUS in each of the 50 ml conical tubes. Do not mix the blood and Ficoll-Paque PLUS. IMPORTANT: To avoid mixing of the cord blood and Ficoll-Paque PLUS, hold the tube at a 45 degree angle and layer the blood mixture slowly.</li>
 <li>Centrifuge at 400 x g for 40 min at 20 &deg;C. Mononuclear cells (MNC) will remain at the plasma-Ficoll-Paque PLUS interface whereas granulocytes and erythrocytes sediment due to higher density at the osmotic pressure of Ficoll-Paque PLUS. Label seven 5 ml round-bottom tubes to be used in Part 3 with the sample ID, date and the following:</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Tube</td>
 <td>Label</td>
 <td>Purpose</td>
 </tr>
 <tr>
 <td>1</td>
 <td>Unstained</td>
 <td>Unstained cells for normalization during flow cytometry</td>
 </tr>
 <tr>
 <td>2</td>
 <td>7AAD</td>
 <td>To determine viability during flow cytometry</td>
 </tr>
 <tr>
 <td>3</td>
 <td>+++</td>
 <td>Contains the cells that will be sorted</td>
 </tr>
 <tr>
 <td>4</td>
 <td>34+ </td>
 <td>To collect CD34+/CD19+ (late pro-B - early pre-BI) cells</td>
 </tr>
 <tr>
 <td>5</td>
 <td>45low</td>
 <td>To collect CD34-/CD19+/CD45low (pre-BI) cells</td>
 </tr>
 <tr>
 <td>6</td>
 <td>45med </td>
 <td>To collect CD34-/CD19+/CD45med (pre-BII) cells</td>
 </tr>
 <tr>
 <td>7</td>
 <td>45high</td>
 <td>To collect CD34-/CD19+/ CD45high (immature B) cells</td>
 </tr>
 </tbody>
</table>
Coat tubes 4, 5, 6, and 7 with 2% FBS and place all tubes on ice.
<ol start="5">
 <li>Aspirate the upper plasma layer carefully and avoid contact with the mononuclear cell layer. Using a 10 ml glass pipette, carefully transfer the mononuclear cell layer to a new 50 ml conical tube. Combine the mononuclear cells from three tubes together into a single 50 ml tube.</li>
 <li>Fill the tube with PBS, mix gently and centrifuge at 300 x g for 10 min at 20 &deg;C. Carefully aspirate the supernatant without disturbing the cell pellet. Repeat 1x. After the first wash, resuspend the pellets and transfer to a single 50 ml conical tube.</li>
 <li>Gently resuspend the cell pellet in 200 &mu;l of PBS. Remove 1 &mu;l of the cell suspension, add it to 1 ml of PBS in a 1.5 ml microcentrifuge tube and set aside for counting (count cells after placing the 50 ml cell suspension in the centrifuge). Fill the tube with PBS and centrifuge at 200 x g for 15 min to remove platelets. Remove the supernatant completely without disturbing the cell pellet.</li>
</ol>
2. Modified B-cell Isolation from Mononuclear Cells Using MACS Separation
<ol>
 <li>Resuspend the pellet from step 1.7 with 160 &mu;l cold (4 &deg;C) EDTA-PBS buffer.</li>
 <li>Add 40 &mu;l B-CLL biotin antibody cocktail. Pipette to mix well and incubate for 10 min at 4 &deg;C.</li>
 <li>Wash cells - add 1 ml cold (4 &deg;C) EDTA-PBS buffer per 10 million cells (cell number determined in step 1.7) and centrifuge at 300 x g for 10 min. Aspirate the supernatant completely and then resuspend the cell pellet in 320 &mu;l cold (4 &deg;C) EDTA-PBS buffer.</li>
 <li>Add 80 &mu;l anti-biotin MicroBeads, mix well and incubate for 15 min at 4 &deg;C.</li>
 <li>Wash cells - add 1 ml cold (4 &deg;C) EDTA-PBS buffer per 10 million cells and centrifuge at 300 x g for 10 min. Resuspend up to 100 million cells in 500 &mu;l of cold (4 &deg;C) EDTA-PBS buffer. Scale buffer volume according to cell number.</li>
 <li>Prepare MACS Columns and MACS Separators during centrifugation (Step 2.5). Place an LS column in the magnetic field of the MACS separator, add 3 ml of cold (4 &deg;C) EDTA-PBS buffer onto the column and allow the buffer to drip through the column. Use a receptacle to catch the flow through. Discard the flow through.</li>
 <li>Place a 50 ml conical tube below the column and pipette the cell suspension onto the column. Allow the unlabeled cells to drip through the column and into the 50 ml conical tube. IMPORTANT: These are the cells that will be used for antibody labeling and cell sorting. Do not discard the flow through. To recover all of the cells from step 2.5, add an additional 1 ml of cold (4 &deg;C) EDTA-PBS buffer to the walls of the conical tube. Mix gently and pipette the remaining cell suspension onto the column. Repeat 1x. Proceed to step 2.8 using the unlabeled cells collected in the conical tube after passing through the column.</li>
 <li>Fill the conical tube containing unlabeled cells with PBS and centrifuge at 300 x g for 10 min. Carefully remove the supernatant without disturbing the cell pellet. Add 200 &mu;l of EDTA-PBS buffer and gently resuspend the cells.</li>
</ol>
3. Antibody Labeling and Preparation for Cell Sorting
<ol>
 <li>Aspirate 1 &mu;l of the cell suspension and place into tube labeled "unstained" (Step 1.4). Add 500 &mu;l EDTA-PBS buffer and store the tube on ice.</li>
 <li>Add 20 &mu;l of each antibody (CD19, CD34 and CD45) per million cells into the remaining cell suspension. Mix well and place the tube in the dark for 30 min at RT. CD antibodies are light sensitive, complete steps 2-4 with the lights turned off.</li>
 <li>After 30 min of incubation with antibodies, aspirate 40 &mu;l of the cell suspension and place it into the tube labeled "7AAD". Add 1 ml PBS into the tube and centrifuge at 500 x g for 2 min. Remove the supernatant and resuspend the cells in 100 &mu;l of binding buffer. Add 7 &mu;l of 7AAD (7-Aminoactinomycin D) and incubate in the dark for 10 min at RT. During the 10 min incubation complete step 3.4.</li>
 <li>Add PBS to the top of the tube containing the remaining cells labeled with CD antibodies. Centrifuge the tube at 500 x g for 3 min. Remove the supernatant, add 500 &mu;l EDTA-PBS buffer and resuspend the cell pellet. Transfer the entire volume of the cell suspension into the tube labeled "+++". To recover all of the cell suspension add an additional 1 ml EDTA-PBS buffer to the 50 ml conical tube and transfer into the tube labeled "+++".</li>
 <li>After incubation (Step 3.3), add 300 &mu;l binding buffer into 7AAD tube. The "unstained", "7AAD" and "+++" samples and the "34 +", "45low", "45med" and "45high" tubes are ready for flow cytometry.</li>
</ol>
4. Cell Sorting Using the MoFlo XDP Flow Cytometer
<ol>
 <li>Set-up MoFlo for sorting: Align lasers; stabilize droplet stream; determine drop delay.</li>
 <li>Prepare single color compensation controls using Invitrogen AbC Mouse bead kit (or similar) according to manufacturer's instructions. Run single color controls, adjusting the voltages of fluorescence channels for optimum separation of positive and negative populations. Set compensation coefficients and apply compensated parameter to collection protocol. Re-establish compensation coefficients each time a new lot of antibody is obtained.</li>
 <li>Create protocol including plots as shown in Figure 2.</li>
 <li>Run unstained sample, set voltage and gain for forward and side scatter, and identify negative fluorescence populations. Because DNA recovery is the goal of the sort, the threshold should be set low (&le;1%) so that DNA-containing debris does not contaminate the recovered samples. *Ensure that the Aerosol Evacuation System is running at all times that live human cells are being run on the MoFlo.</li>
 <li>Run a small aliquot of the +++ sample (~ 50,000 events) to set the gating strategy (Figure 2). Because B-cell progenitors do not scatter identically to mature B-cells, backgate the ungated CD19+/CD34+ population onto the SSC vs FSC plot to ensure the lymphocyte gate includes potential Pro-B cells. From this sample also set the negative fluorescence population for 7AAD.</li>
 <li>Run the 7AAD sample to determine sample viability. Only sort cells from a sample with high viability at the outset (&ge;95%) as dead cells will stain indiscriminately and may contaminate sort populations.</li>
 <li>Set up sort decisions to collect four populations: CD19+/CD34+; CD19+/CD34-/CD45low; CD19+/CD34-/CD45med; and CD19+/CD34-/CD45high. Include the lymphocyte and doublet discrimination gates in the sort decisions of all populations.</li>
 <li>To prevent clogs in the sorting tip, filter +++ sample through a 40 &mu;m cell strainer immediately before sorting and re-filter if any aggregation occurs in sample during sort.</li>
 <li>Sort the cells into collection tubes (Step 1.4) coated with 2% FBS in PBS in a cooled or ice-packed tube holder.</li>
 <li>Immediately after sort is completed, extract DNA.</li>
</ol>

<ol>
	<li>Song, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+tissue-specific+differentially+methylated+regions+(TDMs)+with+differential+gene+expression.">Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression.</a> PNAS. 102 (9), 3336-3341 (2005).</li><li>Ohgane, J., Yagi, S., Shiota, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+The+DNA+methylation+profile+of+tissue-dependent+and+differentially+methylated+regions+in+cells.">Epigenetics: The DNA methylation profile of tissue-dependent and differentially methylated regions in cells.</a> Placenta. 29 (S), 29-35 (2008).</li><li>Brown, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sequential+determination+model+of+hematopoiesis.">The sequential determination model of hematopoiesis.</a> Trends Immunol. 28 (10), 442-448 (2007).</li><li>Clark, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+subsets+in+normal+bone+marrow.">Lymphocyte subsets in normal bone marrow.</a> Blood. 67 (6), 1600-1606 (1986).</li><li>Loken, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+human+bone+marrow.+II.+Normal+B+lymphocyte+development.">Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development.</a> Blood. 70 (5), 1316-1324 (1987).</li><li>Caldwell, C. W., Poje, E., Helikson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+precursors+in+normal+pediatric+bone+marrow.">B-cell precursors in normal pediatric bone marrow.</a> American Journal of Clinical Pathology. 95 (6), 816-823 (1991).</li><li>Tucci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+cord+blood+B+cells+functionally+mature?.">Are cord blood B cells functionally mature?.</a> Clin. Exp. Immunol. 84 (3), 389-394 (1991).</li><li>Ghia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordering+of+human+bone+marrow+B+lymphocyte+precursors+by+single-cell+polymerase+chain+reaction+analyses+of+the+rearrangement+status+of+the+immunoglobulin+H+and+L+chain+gene+loci.">Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci.</a> J. Exp. Med. 184, 2217-2219 (1996).</li><li>Noordzij, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+precursor+B-cell+compartment+in+bone+marrow+from+patients+with+X-linked+agammaglobulinemia+compared+with+healthy+children.">Composition of precursor B-cell compartment in bone marrow from patients with X-linked agammaglobulinemia compared with healthy children.</a> Pediatric Research. 51 (2), 159-168 (2002).</li><li>van Zelm, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ig+gene+rearrangement+steps+are+initiated+in+early+human+precursor+B+cell+subsets+and+correlate+with+specific+transcription+factor+expression.">Ig gene rearrangement steps are initiated in early human precursor B cell subsets and correlate with specific transcription factor expression.</a> J. Immunol. 175 (9), 5912-5922 (2005).</li><li>Rauch, T. A., Pfeifer, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIRA+method+for+DNA+methylation+analysis.">The MIRA method for DNA methylation analysis.</a> Methods Mol. Biol. 507, 65-75 (2009).</li><li>Kanof, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+whole+mononuclear+cells+from+peripheral+blood+and+cord+blood.">Isolation of whole mononuclear cells from peripheral blood and cord blood.</a> Current Protocols in Immunology. , 7.1.1-7.1.7 (1996).</li><li>LeBien, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+human+B-cell+precursors.">Fates of human B-cell precursors.</a> Blood. 96 (1), 9-23 (2000).</li><li>Schmid, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+society+for+analytical+cytology+biosafety+standard+for+sorting+of+unfixed+cells.">International society for analytical cytology biosafety standard for sorting of unfixed cells.</a> Cytometry A. 71 (6), 414-437 (2007).</li><li>Malaspina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+immature/transitional+B+cells+in+HIV-infected+individuals+with+advanced+disease:+correlation+with+increased+IL-7.">Appearance of immature/transitional B cells in HIV-infected individuals with advanced disease: correlation with increased IL-7.</a> PNAS. 103 (7), 2262-2267 (2006).</li></ol>

Authors have nothing to disclose.

The factor with the greatest impact on the success of the protocol is the presence of contaminating debris. If requesting blood from a cord blood bank it is important to have the blood shipped as soon after collection as possible. In addition, samples that are classified as lymphocytosis contain higher numbers of lymphocytes; however, these samples do not have adequate numbers of precursor B-cells and should not be used. To increase the probability of obtaining sufficient numbers of cells for each of the precursor subsets we recommend beginning with at least 85 ml of cord blood.
It is important to note that unlike adult peripheral blood separations, when fractionating umbilical cord blood the mononuclear layer is often contaminated with red blood cells12. This protocol includes an extra wash step to remove contaminating platelets. Protocols describing mononuclear separations from cord blood suggest including a lysis step to remove unwanted red cells. We do not recommend this step because it produces contaminating debris and has a negative impact on the success of the cell sort.
In order to reduce the number of cells that have to be distinguished by flow cytometry it is necessary to perform a B-cell enrichment prior to cell sorting. The protocol provided by Miltenyi Biotec, that accompanies the B-Cell Isolation Kit (B-CLL), has been optimized for peripheral blood and recommends using 10 &mu;l B-CLL biotin antibody cocktail per 10 million cells. This step uses antibodies against CD2 (T cells; NK cells), CD4 (T cells), CD11b (granulocytes; monocytes; macrophages), CD16 (NK cells; macrophages; mast cells), CD36 (platelets), CD235a (erythroid cells) to deplete non-B cells from the umbilical cord blood. It is important to use the kit described and not the B-cell isolation kit II because the former kit contains an antibody against CD43 which is present on pro-B cells. During our optimization of this protocol, we found that a total of 40 &mu;l of B-CLL biotin antibody cocktail is sufficient to produce a positive cell sort result. In addition we found that using as little as 5 &mu;l more of the B-CLL biotin antibody cocktail had adverse effects on the cell sort. Therefore, we highly recommend using 40 &mu;l of B-CLL biotin antibody cocktail for total mononuclear cell numbers between 175-250 million. If starting with lower or higher numbers of cells it may be necessary to scale the reagents accordingly.
There are numerous conflicting publications describing the markers that may be used to distinguish sub-populations of B-cells. Most of the discrepancy can be explained by the fact that differentiation is an on-going process and that the presence or absence of cell surface markers occurs in gradual manner instead of in an all or nothing manner. In this protocol CD34-/CD19+and the intensity level of CD45+ (low, medium, high) expression was used to distinguish pre-BI, pre-BII and immature B-cells with an increase in the level of CD45 expression corresponding to the progression of differentiation6. Pro-B cells have been described by some to be CD19+/CD34+ 13 while others have shown that pro-B cells are CD19-/CD34+ and pre-BI cells are CD19+/CD34+ 9,10. Based on these discrepancies we have designated the CD19+/CD34+cells as late pro-B cells/early pre-BI cells. It is important to note that this strategy was developed to isolate subsets of precursor B-cells that correspond to the cells affected by the disease acute lymphoblastic leukemia. However, there are multiple sorting strategies that may be employed depending on the cell subtype of interest.
Antibody-fluorochrome combinations should be selected based on the capabilities of the available cell sorter. As a general rule, the brightest fluorochrome in your panel should be used to label the least populous antigen and vice versa. In detecting double-stained populations, especially rare events such as these, doublet discrimination (Figure 2B) is a vitally important part of the gating strategy. This ensures that double-positive events are truly single cells stained with both antibodies and not merely two single-stained cells adhered to one another.
High speed, 4-way cell sorting necessarily creates a controlled aerosol environment. Therefore, live human cell sorting should be performed with great care. Published safety and decontamination guidelines are available and should be reviewed and implemented before the sort14. Approval from Institutional Biosafety Committees or their equivalents may be required prior to sorting. For proper decontamination after sorting, bleach should be added to the waste container to a final concentration of 10%. Similarly, all samples tubing as well as all surfaces in the immediate area should be thoroughly decontaminated with a freshly made 10% bleach solution.
In summary, this protocol provides a strategy for obtaining rare populations of precursor B-cells and may be modified to isolate any rare population present in cord blood including hematopoietic stem cells and immature T-cells. Recently, immature B cells have been identified in the peripheral blood of individuals with advanced HIV15. Therefore, the utility of this method extends beyond the study of blood cancers. Finally, we have not yet performed RNA isolations on the flow sorted cells; however, this method should be adaptable with the caveat that during cell sorting the cells should be sorted directly into trizol or an equivalent RNA compatible solution such as RLT from the RNeasy kit available through Qiagen.

In order to identify aberrations that are present in disease, it is vitally important that we use healthy tissues or cells that correspond to the tissue or cell type affected by the disease. One reason for this is that epigenetic variation among tissue types is responsible for regulating gene expression and is critical for cellular differentiation during normal human development1,2. A second reason is that aberrant tissue specific gene regulation may have dire consequences on normal development and is known to contribute to a multitude of disease states including cancer. Therefore, a better understanding of a disease that involves hematopoietic cells requires knowledge of healthy hematopoietic cells.
The development of hematopoietic cells in the bone marrow proceeds through a systematic order of events characterized by changes in the expression of cell surface markers3. Studies involving adult participants have shown that bone marrow usually contains a low number of precursor B-cells4,5; whereas studies involving pediatric participants have shown that the percentage of precursor B-cells is relatively high in individuals less than 5 years of age6. Umbilical cord blood is used as a source of hematopoietic stem cells in the treatment of blood related disorders and malignancies, is readily available via cord blood banks and is enriched for immature B and T cells7 which are the target cells of multiple disorders including leukemia and lymphomas.
Precursor B-cells in the bone marrow have been extensively phenotyped8,9 and can be defined by the presence of specific cell surface markers that can be used to sort these cells into distinct subsets. Normal B-cell differentiation proceeds through a series of stages in the bone marrow beginning with the earliest pro-B cells and culminating in immature or na&iuml;ve B-cells. According to van Zelm and colleagues10, pro-B cells are characterized by the presence of CD34 and in the transition to stage 2 (Pre-BI) CD19 is acquired. Stage 3 (Pre-BII) cells no longer express CD34 and begin to express cytoplasmic IgM. Finally, a defining characteristic of stage 4 (immature B-cells) is the expression of surface IgM. The sorting strategy described in this protocol was first described by Caldwell and colleagues6 and includes the use of only 3 cell surface markers which greatly reduces the complexity and the cost of performing cell sorting experiments. In their work, a relationship between CD45 and the stages of B-cell differentiation was established. They observed that B-cells in the bone marrow display variable levels of expression of CD45. Specifically, cells that expressed high levels of CD45 corresponded to cells that expressed surface IgM (immature B-cells), those that expressed an intermediate level of CD45 corresponded to cells that expressed cytoplasmic IgM (pre-BII cells), and those that expressed low levels of CD45 corresponded to cells that did not express cytoplasmic IgM (pre-BI cells). This protocol uses the strategy developed by Caldwell and colleagues6 to isolate subsets of precursor B-cells from umbilical cord blood (Figure 1) which can be used in downstream assays requiring high quality nucleic acids such as the methylated-CpG island recovery assay (MIRA)11 and quantitative real time PCR assays. The method employs an initial separation using magnetic beads to deplete all non-B cells from umbilical cord blood and requires staining with only 3 antibodies (CD34, CD19 and CD45). The cells that are recovered represent 4 stages of B-cell differentiation: 1) CD34+; CD19+ (late pro-B and early pre-BI); 2) CD34-; CD19+; CD45low (late pre-BI); 3) CD34-; CD19+; CD45med (pre-BII); and 4) CD34-; CD19+; CD45high (immature B-cells).

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DPBS (1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare Bio-Sciences AB</td>
			<td>17-1440-03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MACS Multi Stand</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B-Cell Isolation Kit (B CLL)</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-660</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATLANTA Biologicals</td>
			<td>S11195</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube (1.5 ml)</td>
			<td>MIDSCI</td>
			<td>SS1500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BD Pharmingen 7-AAD (7-Aminoactinomycin D)</td>
			<td>BD Biosciences</td>
			<td>559763</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DB Pharmingen APC Mouse Anti-Human CD19</td>
			<td>BD Biosciences</td>
			<td>555415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DB Pharmingen PE Mouse Anti-Human CD34</td>
			<td>BD Biosciences</td>
			<td>560941</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CD45 FITC</td>
			<td>BD Biosciences</td>
			<td>347463</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BD Falcon 5 ml Polystyrene Round-Bottom Tube</td>
			<td>BD Biosciences</td>
			<td>352058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BD Falcon 50 ml Tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PuraFlow Sheath Fluid, 8X</td>
			<td>Beckman Coulter</td>
			<td>CY30230</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FlowCheck Alignment beads</td>
			<td>Beckman Coulter</td>
			<td>6605359</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultra Rainbow Alignment beads</td>
			<td>Spherotech</td>
			<td>URFP-30-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ViroSafe Aerosol Evacuation Filter</td>
			<td>Beckman Coulter</td>
			<td>ML01330</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ABC Mouse bead kit</td>
			<td>Invitrogen</td>
			<td>A-10344</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>40 &mu;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fisher Scientific Hemocytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>accuSpin Model 3R Benchtop Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>13-100-516</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MoFlo XDP flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>ML99030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aerosol Evacuation Unit</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation of precursor b-cell subsets from umbilical cord blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Between sample variation plays a role in the success of the cell sort (Table 1). The samples with good success rates have low levels of contaminating debris (Figure 3A) and the samples with poor success rates have high levels of contaminating debris (Figure 3B). Between sample variation can be somewhat controlled if the cord blood sample was collected within 24 hr of shipment (O/N first priority).
The flow sorted cells from each precursor B-cell subset are of sufficient quantity and quality to perform nucleic acid isolations. The DNA isolated is of high quality and can be used in downstream analyses. We routinely utilize this DNA in MIRA11 to enrich for methylated DNA (Figure 4).
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Cord Blood Facility Data</td>
 <td>Poor Sort</td>
 <td>Good Sort</td>
 </tr>
 <tr>
 <td>Working Blood Volume with Anticoagulant (ml)</td>
 <td>110</td>
 <td>104</td>
 </tr>
 <tr>
 <td>White Blood Cells [103/ &mu;l]</td>
 <td>8.68</td>
 <td>11.19</td>
 </tr>
 <tr>
 <td>Lymphocytes [103/ &mu;l]</td>
 <td>3.88</td>
 <td>3.76</td>
 </tr>
 <tr>
 <td>Lymphocyte (%)</td>
 <td>44.70</td>
 <td>33.6</td>
 </tr>
 <tr>
 <td>Red Blood Cells [106/ &mu;l]</td>
 <td>3.65</td>
 <td>3.05</td>
 </tr>
 <tr>
 <td colspan="3">In-house Data</td>
 </tr>
 <tr>
 <td>Cell Number after Ficoll-Paque</td>
 <td>206 M</td>
 <td>309 M</td>
 </tr>
 <tr>
 <td>Cell Number after MACS Separation</td>
 <td>22 M</td>
 <td>16.5 M</td>
 </tr>
 <tr>
 <td>Total Events Count (MoFlo XDP)</td>
 <td>30.57 M</td>
 <td>19.97 M</td>
 </tr>
 <tr>
 <td>Viability (%)</td>
 <td>98.00</td>
 <td>98.00</td>
 </tr>
 <tr>
 <td>Cells in Lymphocyte Gate (%)</td>
 <td>17.00</td>
 <td>50.00</td>
 </tr>
 <tr>
 <td colspan="3">CD19+/CD34+</td>
 </tr>
 <tr>
 <td>Total Cell Number</td>
 <td>10959</td>
 <td>48316</td>
 </tr>
 <tr>
 <td>% of Total Events</td>
 <td>0.04</td>
 <td>0.24</td>
 </tr>
 <tr>
 <td>Efficiency</td>
 <td>88%</td>
 <td>88%</td>
 </tr>
 <tr>
 <td colspan="3">CD19+/CD34-/CD45low</td>
 </tr>
 <tr>
 <td>Total Cell Number</td>
 <td>16619</td>
 <td>26941</td>
 </tr>
 <tr>
 <td>% of Total Events</td>
 <td>0.05</td>
 <td>0.13</td>
 </tr>
 <tr>
 <td>Efficiency</td>
 <td>89%</td>
 <td>87%</td>
 </tr>
 <tr>
 <td colspan="3">CD19+/CD34-/CD45med</td>
 </tr>
 <tr>
 <td>Total Cell Number</td>
 <td>469745</td>
 <td>507540</td>
 </tr>
 <tr>
 <td>% of Total Events</td>
 <td>1.54</td>
 <td>2.54</td>
 </tr>
 <tr>
 <td>Efficiency</td>
 <td>89%</td>
 <td>89%</td>
 </tr>
 <tr>
 <td colspan="3">CD19+/CD34-/CD45high</td>
 </tr>
 <tr>
 <td>Total Cell Number</td>
 <td>1896062</td>
 <td>2047142</td>
 </tr>
 <tr>
 <td>% of Total Events</td>
 <td>6.2</td>
 <td>10.25</td>
 </tr>
 <tr>
 <td>Efficiency</td>
 <td>91%</td>
 <td>90%</td>
 </tr>
 </tbody>
</table>
Table 1. Representative cell sort statistics. Rows 1-5 are counts provided by the cord blood facility. The remaining rows are data collected in our laboratory. The poor sort contained high levels of debris and a low percentage of cells in the lymphocyte gate.
<img alt="Figure 1" src="/files/ftp_upload/50402/50402fig1.jpg" /> 
 Figure 1. Flow chart of procedure. Umbilical cord blood is processed and mononuclear cells are isolated using Ficoll-paque plus. An additional wash step is included to remove contaminating platelets. B-cells are isolated from mononuclear cells using the MACS human B-cell isolation kit (B-CLL). This step uses biotin-conjugated monoclonal antibodies (CD2, CD4, CD11b, CD36, Anti-IgE and CD235a) to remove all non-B cells. The recovered B-cells are labeled with CD19-APC, CD34-PE and CD45-FITC antibodies then sorted using the MoFlo XDP flow cytometer to recover precursor B-cell subsets: CD19+/CD34+; CD19+/CD34-/CD45low; CD19+/CD34-/CD45med; and CD19+/CD34-/CD45high.
<img alt="Figure 2" src="/files/ftp_upload/50402/50402fig2.jpg" /> 
 Figure 2. Gating strategy for identifying and sorting populations. Red arrows indicate the gate that is applied to subsequent plot. R4, R5, R6 and R7 gates indicate sorted populations. a) Forward vs. side scatter plot showing enriched lymphocyte population. R1, lymphocyte gate. b) Height vs. width of forward scatter plot for identifying single cells versus doublets. R2, single cell gate. c) CD19-APC positive cells fall into CD34-PE negative and positive populations. R3, CD19+/CD34- gate; R4, CD19+/CD34+ gate indicating desired sort population. d) CD19+/CD34- cells fall into three somewhat distinct CD45-FITC populations. R5 and R6, CD19+/CD34-/CD45low and CD19+/CD34-/CD45med populations, respectively. R7, because of high cell numbers in the CD19+/CD34-/CD45high population, this sort gate encompasses only a portion of the population. All cells of this population do not need to be collected for downstream analyses.
<img alt="Figure 3" src="/files/ftp_upload/50402/50402fig3.jpg" /> 
 Figure 3. a) Low levels of contaminating debris after column enrichment. R1, lymphocyte gate. b) High levels of contaminating debris after column enrichment.
<img alt="Figure 4" src="/files/ftp_upload/50402/50402fig4.jpg" /> 
 Figure 4. Enrichment of methylated DNA from subsets of precursor B-cells. a) Sonicated DNA isolated from precursor B-cell subsets is of high quality. For library construction DNA is sonicated to an average of 200-600 bp. Lane 1: Promega 100 bp ladder (catalog #G2101); Lane 2: Total DNA isolated from CD19+/CD34+ cells; Lane 3: Total DNA isolated from CD19+/CD34-/CD45low cells; Lane 4: 100 ng DNA isolated from CD19+/CD34-/CD45med cells; Lane 5: 100 ng DNA isolated from CD19+/CD34-/CD45high cells. DNA was sonicated using the Diagenode Bioruptor on High for a total of 9 min (30 sec ON; 30 sec OFF). DNA was column purified and visualized on a 1% agarose gel with SYBR green nucleic acid gel stain. b) After MIRA using the ActivMotif MethylCollector Ultra kit, PCR with SLC25A37 (1-6) and APC1 (7-12) is performed to confirm enrichment of methylated DNA. 1&amp;7: Sonicated genomic DNA (no MIRA); 2&amp;8: MIRA-CD19+/CD34+ DNA; 3&amp;9: MIRA-CD19+/CD34-/CD45low DNA; 4&amp;10: MIRA-CD19+/CD34-/CD45med DNA; 5&amp;11: MIRA-CD19+/CD34-/CD45high DNA; 6&amp;12: Water control. Higher amplification of SLC25A37 confirms the enrichment of methylated DNA in the subsets of precursor B-cells.

Watch this Scientific Journal Video about Isolation of Precursor B-cell Subsets from Umbilical Cord Blood at JoVE.com

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

isolation-of-precursor-b-cell-subsets-from-umbilical-cord-blood

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...