Name: Isolation and Transplantation of Hematopoietic Stem Cells (HSCs) (Video) | JoVE Journal
Uploaded: 2007-02-25T00:00:00.000+00:00
Duration: 20 min 38 s
Description: Learn to purify long-term recuperating hematopoietic stem cells and transplant them into mice using sterilization and careful dissection techniques. This method advances stem cell research and therapeutic applications.

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5 minutes

Total bone marrow preparation
<ol>
<li>Four&nbsp;B6 mice are sacrificed and dissected to obtain tibias, femurs, hip and spine.</li>
<li>During the dissections, the limbs and the bones are kept in PBS 2%FCS heat inactivated (optional 2mM EDA - dissection medium).</li>
<li>The clean bones are crushed with mortar and pestle in dissection medium. An efficient way to do it is to crush tibias, femurs and hips of each mouse, then 2 spines at a time. </li>
<li>The cell mixture obtained from each mouse is kept separate and filtered through a 40&mu;m filter into a 50ml falcon tube. In this way, each tube contains the cell mixture obtained out of all the leg bones of a mouse, and half of the mixture obtained from 2 spines. At this stage, the filter is used to separate bone debris.</li>
<li>It is usually advisable to crush the leg bones twice to obtain white (with no bone marrow) bone fragments, and the spine fragments 2 or 3 times.</li>
<li>Fill up the tubes to have them balanced, and spin 5 min 1200rpm.</li>
<li>Aspirate the supernatant and loosen the pellet by dragging the tubes on the tube rack (this step is important after each centrifugation to minimize clump formation and cell loss). Resuspend each tube in 1 ml PBS 2% FCS (NO EDTA from now on) if you are doing lineage depletion, or in whatever volume is otherwise convenient for you.</li>
</ol>
Lineage depletion
<ol>
<li>This procedure allows you to eliminate highly differentiated cells and obtain a very heterogeneous population of cells, enriched for stem and progenitor cells. If you are sorting HSC, this step drastically reduces the number of cells that have to go through the cell sorting, therefore reducing sorting time and allowing you to sort HSC out of a higher number of mice, so that you can obtain a higher number of HSC in one single sorting.</li>
<li>If you are going to sort later, take out 50&mu;l of cell mixture (I usually take a bit from each tube) for sorting controls (unstained and Sca1, c-Kit, CD48, Flk2 single color controls). </li>
<li>Add Lin cocktail 30ml/tube. Lineage cocktail contains a variety of antibodies, which unfortunately changes a little from&nbsp;lab to lab. In the Scadden lab, we use biotinilated antibodies against Gr1, Ter119, CD4, CD8, CD3, B220. Their final dilution in the staining is 1:700. </li>
<li>Vortex quickly to mix, and incubate for 15 min at 4&deg;C. </li>
<li>During the incubation, prepare the columns on the magnets and 15ml elution tubes under the columns. Degas some PBS using a steriflip filter (you should see little air bubbles moving towards the surface of the PBS due to vacuum aspiration), and equilibrate the columns by applying 3ml degassed PBS to each column. </li>
<li>The column flow is about 1ml/5min, so it takes about 15 min to have the columns ready to use. </li>
<li>At the end of the incubation, add PBS 2% FCS to fill the tubes and spin down for 5 min at 1200rpm. </li>
<li>Aspirate the supernatant, loosen the pellets and resuspend in 1 ml/tube degassed PBS. </li>
<li>If you are sorting later, take out total bone marrow Lineage single color staining ctrl (15ml, a bit from each tube). </li>
<li>Add streptavidin beads 30ml/tube, vortex quickly to mix, and incubate for 15 min at 4&deg;C. </li>
<li>At the end of the incubation, add 2 ml/tube of degassed PBS. Put new collection tubes under the columns, and apply each suspension to a column, filtering through a 40&mu;m filter. </li>
<li>Add another 3 ml of degassed PBS to each tube to wash it. Once the columns are empty, apply again using the same 40&mu;m filter. </li>
<li>The eluate contains the lineage depleted cell mixture, a heterogeneous population enriched for stem and progenitors cells. Pool the content of the 4 tubes into 2 tubes. Spin for 5 min at 1500rpm. </li>
<li>Aspirate supernatant. The pellet should be white or mainly white. Loosen the pellets and resuspend them together in 1ml total PBS 2% FCS if you are sorting later, otherwise resuspend in whatever volume is convenient for you. </li>
</ol>
LKS or long term HSC sorting
<ol>
<li>Take out lineage depleted single color staining ctrl (15ml). This will allow you to assess the efficiency of your lineage depletion.</li>
<li>The cell mixture to sort is stained in the 15ml tube.</li>
</ol>
Add antibodies:

<table border="0">
<tbody>
<tr>
<td>Sca PE-Cy5.5</td>
<td>1: 100</td>
<td>10ml</td>
</tr>
<tr>
<td>Kit APC</td>
<td>1:100</td>
<td>10ml</td>
</tr>
<tr>
<td>SA PE-Cy7</td>
<td>1:500</td>
<td>2ml</td>
</tr>
</tbody>
</table>

To sort long term HSC, aslo&nbsp;add:

<table border="0">
<tbody>
<tr>
<td class="jove_content">CD48 FITC</td>
<td class="jove_content">1:1000</td>
<td class="jove_content">1ml</td>
</tr>
<tr>
<td class="jove_content">Flk2 PE</td>
<td class="jove_content">1:200</td>
<td class="jove_content">5ml</td>
</tr>
</tbody>
</table>

Also, prepare single staining controls using the total bone marrow kept aside after the dissection and the total bone marrow lineage stained. These stainings can be done directly into FACS tubes.
Make sure to be in the dark!
<ol>
<li>Vortex quickly to mix and incubate at 4&deg;C for 20 to 30 minutes, re-mixing half way through.</li>
<li>To wash away the excess of antibodies, fill up the tubes with PBS 2% FCS and spin for 5 min at 1500 rpm.</li>
<li>Remove supernatant, resuspend all pellets and move them to new FACS tubes with filter caps. It might take a while to get the cells to go through these filters, and it is important to apply some more PBS afterwards to wash the filter and minimize cell loss. This step is necessary to avoid clogging of the sorter, which is the worst nightmare of every cell sorter user.</li>
<li>When handing out your cells to the cell sorting facility, make sure to also prepare a tube with PBS 10% HI FCS where the cells will be collected.</li>
<li>If you are sorting LKS cells, you will obtain a heterogeneous population containing long term and short term repopulating HSC and multipotent progenitors. An average yield is 300,000 LKS from 4 mice.</li>
<li>If you have also used CD48 and Flk2 antibodies, you can separate each population of long term HSC, short term HSC and MPP. LT-HSC are the most rare population. An average yield is about 50,000 to 60,000 cells from 4 mice.</li>
</ol>

isolation and transplantation of hematopoietic stem cells (hscs)

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

isolation-and-transplantation-of-hematopoietic-stem-cells-hscs

Research

JoVE Journal

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Learn to purify long-term recuperating hematopoietic stem cells and transplant them into mice using sterilization and careful dissection techniques. This method advances stem cell research and therapeutic applications.

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

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