EXPERIMENTS WITH PERIPHERAL BLOOD
STEM CELLS
By
Francis Wodie
Dr. David Prentice Life Science 692
Department of Life Science Summer I 2003
Indiana State University
INTRODUCTION:
Adult stem cells, which come from different tissues of the body, have promising properties that make them unique and very promising for clinical use. They can continue to divide and maintain a pool of stem cells, and once given the correct signal they can differentiate into tissues including endodermal cells, mesodermal cells and ectodermal cells, independently of where they came from. Adult stem cells also show an ability to home in on damaged tissues, and a patient’s own adult stem cells can be used for treatment, preventing problems of transplant rejection. Based on these properties, these cells are studied and already used in the treatment of degenerative diseases. Already bone marrow stem cells have been transformed into nerve tissues, insulin-secreting cells, liver tissue, and various other body tissues.
For this experiment the ultimate goal is to try to transform peripheral blood stem cells into nerve. We conducted 2 experiments, to obtain some basic culture parameters for these human cells. In the first experiment we were trying to determine what is the best concentration of serum to use, and in the second how often we need to feed the cells.
For the current information regarding adult stem cells, see the extensive review written by Dr David Prentice, that can be downloaded from the following address: http://bioethics.gov/background/prentice_paper.html
METHODS and EXPERIMENTS:
In experiment 1, we followed what we labeled as blood stem cell isolation protocol number 1.
On June 10th 2003, we collected with the assistance of Dr Marcella Stevens, 3 tubes of peripheral blood from Francis Wodie or a total volume of 14 ml that we put in a 50ml tube.
We then added 14 ml of DMEM+2% FBS to the blood.
In another 50ml tube, we put 15 ml of histopaque.
We added the diluted blood on top.
We centrifuged for 20 minutes at 1200xg, at room temperature.
We removed the plasma and discarded it. The buffy coat went to a 15ml tube.
We subdivided the buffy coat into 3 15ml tubes and added 10 ml of DMEM+2% FBS to each tube.
We centrifuged for 5 minutes at 800xg, at room temperature. Then, we removed the supernatant and discarded it.
We resuspended each pellet into 10 ml of DMEM+2% FBS.
We centrifuged for 5 minutes at 800xg, at room temperature to wash the cells, and we removed the supernatant.
Finally, the pellet of one of the tubes was resuspended into 13 ml of DMEM+5% FBS. The pellet of another tube was resuspended into 13 ml of DMEM+10% FBS.
The pellet of the last tube was resuspended into 13 ml of DMEM+20% FBS.
We added 2ml of these mediums to 6 35mm plates, or a total of 18 plates.
With the left over from the tubes, we did a cell count.
On June 11th, we removed the medium from 2 of the plates with 5% FBS. We did the same with 2 of the plates with 10% FBS and 2 of the plates with 20% FBS.
We then added 2 ml of PBSA to each plate and we removed it.
We added 1 ml of trypsin to each plate for 60 seconds. We removed it.
Finally we added 1 ml of PBSA to each tubes and scraped the plates when necessary.
We did a cell count using the hemacytometer.
On June 13th, we repeated the exact same protocol and did another cell count.
We then removed the medium from the 6 plates that were left and added fresh medium, with the same concentration of serum each plate had before.
On June 15th, we repeated the protocol used the 11th and the 13th, and we had a cell count for the last 6 plates.
For experiment 2, we also followed the blood stem cell isolation protocol.
On June 17th, we collected 2 tubes of peripheral blood from Francis Wodie. The total volume of blood was 8 ml that we put in a 50ml tube. We added 8 ml of DMEM+2% FBS to the tube.
In another 50 ml tube we put 15 ml of histopaque, and we added the diluted blood on top.
We centrifuged for 20 minutes at 1200xg at room temperature. We removed the plasma.
We also removed the buffy coat and subdivided it into 2 15ml tubes.
We added 10 ml of DMEM+2% FBS to each tube.
We centrifuged for 5 minutes at 800xg at room temperature. We removed the supernatant.
We resuspended the pellet into 10 ml of DMEM+2% FBS.
We centrifuged for 5 minutes at 800xg at room temperature. We removed the supernatant.
We resuspended the pellet of each tube into 9 ml of DMEM+10% FBS.
We added 2 ml of medium to each 35mm plate we had or a total of 8 plates.
With what was left over in the tubes, we did a cell count.
The 8plates were divided into 2 categories: the plates fed once a week and the plates fed twice a week.
On June 20th, we removed the medium from all the plates and added fresh medium.
On June 23rd, we picked one plate from each category.
We removed the medium from these 2 plates, added 2 ml of PBSA to each one and removed it.
We then added 1 ml of trypsin to each plate for 60 seconds and removed it.
We finally added 1 ml of PBSA to each plate, scraped and did a cell count.
On June 24th, we only re-fed the plates that were supposed to be fed twice a week.
On June 27th, we re-fed all the plates.
On June 29th, we removed the medium from the plates that were left.
We added 2 ml of PBSA to each one and removed it.
We added 1 ml of trypsin to each one for 60 seconds and removed it.
We added 1 ml of PBSA to each plate, scraped and did a cell count.
On July 24th, following Dr Prentice’s advice, we decided to do a duplicate of experiment 1. Therefore, we followed the exact same procedure.
Day 0 was July 24th. This day we collected a total volume of peripheral blood of 13 ml
Day 1 was July 25th.
Day 3 was July 27th. On this day we did not only have a cell count, but like in the first experiment, we also re-fed the plates that were destined to be used on day 5.
Day 5 was July 29th.
RESULTS:
Experiment 1
|
Days |
0 |
1 |
3 |
5 |
|
DMEM+5%FBS (X 104cells/ml) |
1.5 |
1.75 2.25 |
3 2.25 |
2.25 3.25 |
|
DMEM+10%FBS (X 104cells/ml) |
1 |
0.75 3.75 |
2.75 3.5 |
3.25 3.5 |
|
DMEM+20%FBS (X 104cells/ml) |
1.75 |
1.25 0.25 |
4.75 4 |
5.25 5.5 |
Duplicate Results:
|
Days |
0 |
1 |
3 |
5 |
|
DMEM+5%FBS (X 104cells/ml) |
1.5 |
1.5 1 |
5.5 2.75 |
3 3.75 |
|
DMEM+10%FBS (X 104cells/ml) |
1 |
1.25 1.5 |
4.75 5.75 |
5.25 5.5 |
|
DMEM+20%FBS (X 104cells/ml) |
1.75 |
1 0 |
5.5 6.25 |
5.5 6 |
Average of the results (cell numbers X 104cells/ml):
|
Days |
0 |
1 |
3 |
5 |
|
DMEM+5%FBS |
1.5 |
1.625 |
3.375 |
3.062 |
|
DMEM+10%FBS |
1 |
1.812 |
4.187 |
4.375 |
|
DMEM+20%FBS |
1.75 |
0.833 |
5.125 |
7.41 |
Based on the graph obtained with the average of the cell counts, we can say that in this experiment, when using DMEM+5% FBS, we have an increase in the cell count from day 1 to 3, followed by a leveling off or slight drop from day 3 to 5. When using DMEM+10% FBS, we notice a similar increase, though slightly better, from day 1 to 3. There is a slight increase from day 3 to 5. When using DMEM+20% FBS, the cell count rises from day 1 to 3 in similar manner than the two previous categories, though the final number is superior. The rise continues from day 3 to 5.
Experiment 1’
|
Days |
0 |
1 |
3 |
5 |
|
DMEM+5%FBS (X 104cells/ml) |
60 53.25 |
10.5 12 |
14.75 11.5 |
3 2.25 |
|
DMEM+10%FBS (X 104cells/ml) |
48.25 56 |
5.5 4.5 |
8.5 7.75 |
3.75 5.75 |
|
DMEM+20%FBS (X 104cells/ml) |
51.5 47.75 |
10.5 8.5 |
21 27.75 |
7.25 8 |
Duplicate Results:
|
Days |
0 |
1 |
3 |
5 |
|
DMEM+5%FBS (X 104cells/ml) |
60 53.25 |
8.75 8.5 |
9 8.25 |
3.5 3.75 |
|
DMEM+10%FBS (X 104cells/ml) |
48.25 56 |
9.5 10.25 |
12.75 9.5 |
2.75 3.25 |
|
DMEM+20%FBS (X 104cells/ml) |
51.5 47.75 |
7.75 9 |
7.75 11.25 |
9.75 9 |
Average of the results (cell numbers X 104cells/ml):
|
Days |
0 |
1 |
3 |
5 |
|
DMEM+5%FBS |
56.625 |
9.937 |
10.875 |
3.125 |
|
DMEM+10%FBS |
52.125 |
7.437 |
9.625 |
3.875 |
|
DMEM+20%FBS |
49.625 |
8.937 |
16.875 |
8.5 |
In the duplicate experiment, no matter what the concentration of serum was, the cell counts followed the same path: a rise from day 1 to day 3, followed by a drop from day 3 to day 5. The numbers are better when using 20% FBS. When using 10% FBS we obtain a slightly better increase than with 5% FBS from day 1 to day 3, but from at day 5, the cell count is higher with 5% FBS.
Experiment 2
|
Days |
1 |
6 |
12 |
|
Cells fed 1X a week (X 104cells/ml) |
52; 45 |
5.75; 4 |
4; 4; 3.75; 5.75 |
|
Cells fed 2X a week (X 104cells/ml) |
48; 53 |
2.5; 3.25 |
5; 5.25 |
Average of the results
|
Days |
1 |
6 |
12 |
|
Fed once a week (X 104cells/ml) |
48.5 |
4.87 |
4.37 |
|
Fed twice a week (X 104cells/ml) |
50.5 |
2.87 |
5.12 |
The graph shows us that after 6 days, the cells fed once a week were more important in number than the cells fed twice a week. 6 days later, the cells fed twice a week were more important in number that the cells fed once a week. The cell count of the cells fed twice a week rose, but the cell count of those fed once a week slightly decreased.
DISCUSSION:
We did not consider the cell counts obtained on the different day 0, because it is our belief many of the cells counted on day 0 were regular blood cells and not blood stem cells. Only after we plated the cells were we able to keep the stem cells wanted.
The first experiment showed us that although it is safe to use the DMEM+10% FBS, the DMEM+20% FBS is better. We had confirmation of these findings in the second experiment. Although the cell counts number dropped from day 3 to 5, the higher numbers were obtained with DMEM+20% FBS. If we were to recommend a serum concentration for future experiments, we would probably recommend a DMEM+20% FBS or DMEM+15% FBS.
We can add that although the numbers were higher in the duplicate experiment, the cell count numbers obtained on day 5 are somewhat similar to those obtained in the original experiment.
The second experiment showed us that it is better to feed the cells twice a week. If we were to recommend one of the conditions we tested for future experiments, we would recommend to feed the cells twice a week. We would also recommend to duplicate the experiment if time and supplies allow to do so.
Tags: biology, experiment, francis wodie, Indiana State University, life sciences, peripheral blood, stem cell, stem cells, wodie
