The Differentiation of Hematopoietic Stem CellsRyan JahansoozAssisted by Tyler Voss, Zac Foster, and Hunter Wolfbear DrobenaireBio 001 Lab Section 16 10-16-13Abstract:This lab, while being the opposite of quick and painless, made up for its complexity with the huge amount of knowledge that was gained from it. It began with the preparation of a slide with cells. The slide was prepared using a centrifuge, a special slide setup, and a sample of cells suspended in a solution. Once the slide was prepared, it and three other slides were dipped into variety of treatments tosee how each treatment affected the cells. This also allowed the viewer to see the cells more clearly using a light microscope. The number of each type of cell was recorded, and the stations were cleaned.Introduction:Stem cells are the cells that all the different varieties of cells in our bodies “stem” from. They are undifferentiated, and thus are able to adapt to whichever job is needed by differentiating themselves. This adaptability makes them extremely useful when it comes to healing and regenerating from injuries, but they also have a major role in the creation of fetuses in the womb. These two types of stem cells are called pluripotent and multipotent. The original stem cell that was created by a conjoining sperm and egg cell is considered to be totipotent.Stem cells differentiate into a number of different cells. The cell types used in this experiment are known as erythrocytes( red blood cells), macrophages(a type of white blood cell), or neutrophils(another type of white blood cell). Each of these is absolutely essential for life as they provide the service of transporting resources around the body as well as fending off attacks from viruses and other infectious invaders. It is important that each of these cells be able to differentiate to adapt to other jobs because they work closely with one another in our bodies environment. The overall goal of the lab is to learn how cytokines influence which immature hematopoietic cells adapt into one of the three types of cell stated earlier. To study this, immature hematopoietic cells will be put through a series of cellular straining and microscopy techniques to discover how exposure to cytokines directs them to differentiation.Methods and Materials:The experimental procedures for this lab were adapted from a previously supplied protocol (Dulai, 2012). Any deviations from lab protocol that happened were due to contamination from the slides being in the same vials. Minor crossing of the two cytokines may have occurred when small traces of the chemicals left in the distilled water were transferred to the other vials. It is unlikely that this had any noticeable effect on the labs.Results:From the careful observation of over two hundred cells, with approximately fifty from each treatment group, it became obvious that each treatment yielded different results. Group one exhibited near 100% undifferentiated cells which were all monocytes (FIG 1). Group two was mostly composed of erythrocytes, with a smaller amount of neutrophils, and almost no macrophages or undifferentiated cells(FIG 2). Group 3 contained large amounts of neutrophages followed by decent numbers of macrophages and almost no erythrocytes (FIG 3). The last group seemed to be much stronger than group two, in that almost all of the cells were erythrocytes with smaller traces of neutrophils and macrophages (FIG 4). All data can be seen together comparatively in FIG 5 and FIG 6. Each graph simply displays the numbers in tables 1-4 visually.FIG 1Treatment GroupStaining Method Number of NeutrophilsNumber of ErythrocytesNumber of MacrophagesNumber of Undifferentiated CellsTotal Cells Counted1 May-Grunwald/Giesma0 0 0 50 50o-dianisidine/May-Grunwald/Glemsa0 0 0 50 50FIG 2Treatment GroupStaining Method Number of NeutrophilsNumber of ErythrocytesNumber of MacrophagesNumber of Undifferentiated CellsTotal Cells Counted2 May-Grunwald/Giesma13 23 14 7 57o-dianisidine/May-Grunwald/Glemsa21 26 3 13 63FIG 3Treatment GroupStaining Method Number of NeutrophilsNumber of ErythrocytesNumber of MacrophagesNumber of Undifferentiated CellsTotal Cells Counted1 May-Grunwald/Giesma23 0 27 12 62o-dianisidine/May-Grunwald/Glemsa23 12 15 16 62FIG 4Treatment GroupStaining Method Number of NeutrophilsNumber of ErythrocytesNumber of MacrophagesNumber of Undifferentiated CellsTotal Cells Counted1 May-Grunwald/Giesma1 42 7 7 57o-dianisidine/May-Grunwald/Giemsa18 19 13 18 68Fig 5: May-Grunwald/Giesma Statistics1 2 3 4010203040506070NeutrophilsErythrocytesMacrophagesUndiff. CellsTotalFig 6: Odianisidine/May-Grunwald/Giesma1 2 3 401020304050607080NeutrophilsErythrocytesMacrophagesUndiff. CellsTotalDiscussion:From figs 2 and 4, it is clear that group treatments 2 and 4 were by far the most effective at influencing stem cell commitment to the erythroid lineage. You can see that this is true because those treatment groups show far greater numbers of erythrocytes, far more than groups one or three. Overall, it appears as though group four is the leader and thus is the go-to treatment if the goal is to force stem cells to differentiate to the erythroid lineage.From fig 3, it is clear that group treatment three was the most effective at influencing stem cell differentiation to the neutrophil lineage. You can see that this is the case because this group shows the highest differentiation rate, though groups two and four still changed a few.It also appears that group three is also the leader in differentiating stem cells to the macrophage lineage. You can see this by observing fig 3. It is clear that this is the case because this group shows the highest differentiation rate.And from figs five and six, it appears that each treatment group can be used to create specific differentiations in stem cells. Groups four and two are the best for procuring erythrocytes, while group three is the best for creating neutrophils and macrophages. Group one wins an award for participation, as it did not differentiate any stem cells. It does not appear that any treatment produced 100% of any type of cell; however, it would appear that specific cytokine combinations did differentiate specific differentiations. Conclusion:In this lab, it became abundantly clear how some chemicals called cytokines can be used to influence how stem cells differentiate. Some cytokines excel at creating specific lineages while others cancreate many of different types. This knowledge of