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The Center for Translational Research in the Lung (CTRLung) have shown that the transfer of bone marrow derived mesenchymal stem cells (BMDMSC)
decreases bleomycin-induced lung injury and fibrosis in mice. Several recent studies have demonstrated that stem cells derived from bone marrow can be
recruited to the injured lungs and participate in both lung repair and injury. The chemokines, signaling chemicals that initiate cell migration, involved in this
migration of BMDMSC are not fully understood. Past hypotheses concerning the effect of aging upon this migration have attributed decreased migration to
a decrease in the number and production of BMDMSC. In this study we hypothesized that there is decreased migration of BMDMSC with age, but that this
decrease is due to a decrease in the BMDMSCs ability to respond to chemokines that occurs with aging. To determine the ability of BMDSC to migrate in
response to chemokines an in vitro model was used comparing the migration of stem cells from SAMP and SAMR mice. The chemokines studied included
SDF-1, IL-17, CXCL2 and CXCL3. IL-2 was used as a negative control. SDF-1 is a ligand thought to be responsible for stem cell recruitment. IL-17 is has been
hypothesized to have a similar effect. CXCL2 and CXCL3 are thought to be chemokines that may be produced by cancer tumors to attract stem cells and then
use the stem cells for angiogenesis and to further their own growth. Parallel studies of Female SAMP8 mice, 11 months of age and Female SAMR mice, 11 month
of age were performed. Bone marrow was harvested from the mice, and stem cells from that bone marrow were used. A migration assay was performed that
examined the chemoattractant effects of the aforementioned chemokines. Upon comparison of the SAMP and SAMR mice, it was found that the difference between
the number of cells migrating in response to the chemokines in the two strains was statistically significant only for CXCL3 (p<0.05). The combination of SDF-1
& IL-17 yielded a p-value of 0.386, which was the next closet chemokine to showing a statistically significant difference between the two strains in response to
chemokines. Thus, aging does have an effect on the ability of cells to migrate in response to CXCL3 and possibly a combination of SDF-1 & IL-17 as well as
the others. Thus, the decreased migration of stem cells with aging can be attributed to a reduced ability to respond to chemokines rather than a decrease in the
number of stem cells. However, more migration assays are needed to determine the efficacy of the cytokines.
Many current advances in medicine are providing new possibilities for diminishing the aging process and increasing the average lifespan. Mesenchymal
stem cells are a new potential therapy for protection of organ tissue, with an enormous ability for limiting or reversing injuries to aged tissue or degeneration
of tissue due to ageing itself. In this study we used Senescence Accelerated Mice. There are two main strains of these mice: SAMP, for senescence-prone,
and SAMR, for senescence-resistant. The SAMP mice have a 26% shorter lifespan. The Senescence Accelerated mouse provides an experimental model in
which the mice undergo accelerated senescence and display most of the common age-associated disorders seen in aging humans.
Parallel studies of the SAMP and SAMR mice were conducted. Specimens from each groups, aged 11 months were euthanized by isofluorane inhalation and
cervical dislocation and bone marrow was harvested from the femur. Inflated lung samples were taken and were examined histologically using H&E staining.
The in vitro testing of responses of both whole bone marrow-derived cell suspensions and bone marrow-derived mesenchymal stem cells will be done by
determining their chemotactic responses to chemokines: SDF-1, IL-17, CXCL2, CXCL3 and the negative control IL-2. The bone marrow-derived cell suspensions
were directly from the bone marrow and required no sorting. However, in order to obtain bone marrow-derived mesenchymal stem cells, the bone marrow-derived
cell suspension, after performing the migration assay, were cultured for 48 hours in Messencult with serum. Upon 48 hours the mesenchymal cells were gathered
by washing the cells with PBS to remove the cells that are not mesenchymal stem cells. Next, trypsin is used to detach the cells from the plate. After obtaining
mesenchymal stem cells a second migration assay would be performed; this assay with only mesenchymal stem cells, which is in contrast to using bone marrow
cell suspensions in the first assay. Each set of two assays comprises 1 experiment. The migration assay was comprised of a 24-well plate and 24 collagen
coated inserts to allow for stem cell migration (Figure 7). 12 wells were used in each individual assay. 6 wells and inserts for the SAMP group and 6 wells and
inserts for the SAMR group. Each well would contain 600μl medium without serum, and 3μl of the designated chemokine or control. The insert would contain
exactly 200,000 cells in medium without serum. The inserts are placed in their designated wells and placed in a 37 degree Celsius incubator for 3 hours. After
incubation the solution in the well is evaluated for migrated cells using a cytometer.
The results of this study indicate that the changes in stem cell migration associated with aging may be due to a decreased ability to respond to chemokines.
CXCL3 was the only chemokine that showed a statistically significant difference between the two strains in migration cells. More assays are needed to study the
effects of the other chemokines in this study more in depth. It is possible that more of the tested cytokines could show statistically significant differences between
the SAMP and SAMR mice if more assays are complete so that we have a larger number and consequently a smaller standard deviation. Also, this study supports
that SDF-1, IL-17, CXCL2 and CXCL3 are effective chemokines. Also, SDF-1 and IL-17 may not work synergistically, evidenced by the decrease in migration
when combined in comparison to the migration occuring in response to SDF-1 alone. The possible chemoattractant properties of IL-17 have not been studied
previously. Currently IL-17 is accepted as a cytokine that promotes proliferation of hematopoetic progenitors. It should also be noted that there were differences
in the results of the whole bone marrow cell migration assays and those using only mesenchymal stem cells. This presents information that could lead to a future
study of the differences between hematopoetic and mesenchymal stem cell activity and sensitivity associated with aging.
Decreases in stem cell migration do occur with aging and the mechanism of these changes is the ability of bone marrow-dervied stem cells to respond to
chemokines. In the future more trials of the assay are necessary to support statisical significance. Also, a study comparing hematopeoptic and mesenchymal
stem cell physiology in relation to aging would provide new information.
Special thanks to Smita Iyer, Edilson Torres, Dr. Ana Mora, and Robert Joodi. Funding provided by the Center for Translational Research in the Lung and
The Howard Hughes Medical Institute Grant #52003727.
When lungs become injured the lung send out chemical messages, called chemokines, to the bone marrow to recruit stem cells and cause the stem cells to
move to the lungs to repair the injury. As a person gets older, the amount of stem cells moving to the lungs after receiving the chemical messages decreases.
Some people believe that this decrease in stem cells moving to the lungs is due to a decreased number of stem cells that comes with age. However, it is
hypothesized in this study that the reason for the decrease is the stem cells’ decreased ability to respond to the chemical messages.
To study this, two groups of mice were used. One group aged more easily, while the other was resistant to aging. We used the same number of stem cells in
the study for both groups and the same amounts of chemical messages so that we could test the ability of the stem cells to respond to the chemical message.
It was found that for some of the chemical messages, the mice that were resistant to aging had more stem cells migrating than the mice that were prone to aging.
More trials are needed in order to be more confident in our results.
Cell Culture, Chemotaxis Cell Assaysm, H&E Histology, Paraffin Sectioning, Bone Marrow Harvesting
Stem Cells, Aging, IL-17, CXCL2, Lung Injury
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