Wound Recovery with Hematopoietic Stem Cells in scid and WT Mice at

Different Ages

Aim: Apply CD34+ cells to the wounds of non-leaky scid RAG-1 deficient mice and wild type
mice belonging to two different age groups and measure any differences in healing rates as
compared to controls.

Strategy

If the immune system fails to distinguish between cancer stem cells and healthy stem
cells in old age, then mice with severe combined immune deficiency (SCID) would be
expected to recover from a wound healed by young hematopoietic stem cells more quickly
than wild type (wt) mice treated with the same stem cells. The general experimental setup
will involve wounding mice of two different age groups, and determining if there is a difference
in wound healing rates among scid, wt, stem cell treated, and non-stem cell treated mice.
The rate of wound healing will serve as an approximate quantitative measure of the aging
process, which results in a decreasing ability to repair damage over time. The observation
that the stem cell treated scid old mice do not heal significantly faster than stem cell treated
wt old mice would suggest that the proposed hypothesis is incorrect. In other words, this
observation would suggest that the immune system does not inhibit the healing process and
is not failing to distinguish between cancer stem cells and healthy stem cells in old age.
There are several different types of immunodeficient mice which have been studied.
The first immunodeficient mouse was identified in 1983 and developed from a spontaneous
scid mutation which resulted in a mutated DNA-PKcs enzyme [1]. This mutation causes an
inability to properly rearrange V(D)J genes. Such rearrangement is necessary for valid
antibody and TCR (T cell receptor) development. Without properly rearranged antibodies and
TCR, mature B and T cells are absent. These mice exhibited approximately normal lifespans
in pathogen free environments, but developed more lymphomas than normal [1]. These mice
were also considered “leaky” because some functional antibodies and TCRs could be
produced occasionally despite the mutated DNA-PKcs enzyme [2]. Researchers were later
able to develop “non-leaky” scid mice by knocking out the RAG-2 gene, which resulted in an
inability to initiate V(D)J rearrangement [3]. Humans with mutated RAG-2 genes have been
found to occasionally develop problems such as Omenn syndrome and granulomatous
inflammation in addition to their immunodeficiency, but researchers have stated that the
produced RAG-2 deficient mice appear normal [4]. Therefore, for this experiment, we will
choose to use the RAG-2 mouse as our “non-leaky” scid mouse model, although many other
immunodeficient strains have also been identified such as CH3-scid [5], NOD/scid [6], beige,
and RAG-1 [7].
The general experimental strategy will be to treat wounded RAG-2 scid and WT mouse
groups with hematopoietic stem cells. The hematopoietic stem cells used will be CD34+ cells
which have also been used to heal the wounds of NOD/scid mice which are immune deficient
diabetic models [6]. This is possible because adult stem cells extracted from blood can
differentiate into a variety of cell types including endothelial cells [8]. CD34+ and CD34- will
be applied to eight different groups of mice as follows: CD34+ scid young, CD34+ wt young,
CD34- scid young, CD34- wt young, CD34+ scid old, CD34+ wt old, CD34- scid old, and
CD34- wt old. A young group of wt mice that are not wounded will be used as a source of
CD34+ cells.
Methodology

The mice for this experiment will be obtained from Jackson laboratories. The scid mice
will be produced by breeding CbyJ.B6(Cg)-Rag2tm1Cgn/J with the Cre recombinase strain
C57BL/6J-Tg(Mx1-cre)1Cgn/J as described by the vendor. BALB/cByJ mice will be used as
the wt mice and are the recommended controls for the scid mice chosen. 6 month old mice
will be used for “young” mice groups, and 15 month old mice will be used for “old” mice
groups. A two-factor factorial ANOVA with p<0.05 will be used to compare the wound healing
results between matched groups throughout the analysis.
CD34+ cells will be purified from the blood of young wt mice. PBMCs (peripheral blood
mononuclear cells) will be collected from the blood, which will then be centrifuged to separate
the PBMCs from red blood cells as described by Sivan-Loukianova et al [6]. CD34+ antibody
coated magnetic beads will be used to isolate CD34+ cells. These cells will then be released
with enzymatic digestion [9]. The remaining CD34- cells will be used with the control groups.
All groups of mice with the exception of the CD34+ donor group will be wounded. The
mice will first be anesthetized with ketamine and xylazine. The skin will then be cleaned with
povidone-iodine, and a 4 mm wound will be inflicted on the skin of the back. Three wounds
per mouse will be inflicted. The CD34+ groups will receive these cells via an injection of the
purified CD34+ cells just below the bed of the wound. A similar procedure will be performed
with the CD34- cells for the negative control groups.
The rate that the wounds heal will be determined by measuring the size of the wound
over time. One of the wounds from each mouse will be harvested 7, 14, or 21 days after
healing as described by Sivan-Loukianova et al [6]. The tissues will be hematoxylin and eosin
stained and analyzed with a microscope such as the Nikon E600. The area of the wound,
number of blood vessels, and total area of blood vessels will all be determined and used in
analysis to compare the rates of healing among the different groups. In addition to harvesting
the tissues at certain time periods, images of the wounds will be acquired on a daily basis to
gather subtle changes in wound size over time.
These experiments could be performed in approximately 3 months as outlined in the
timeline (Figure 1).

Figure 1: Timeline of procedures for the experiment.

Interpretation of Results

If the proposed acquired autoimmune stem cell hypothesis is correct, then several
predictions can be made. For example, the scid mice would be expected to heal more quickly
than the wt mice. The proposed hypothesis does not suggest that immunity against stem
cells is the only cause of aging. Therefore, older scid mice would still be expected to heal
more slowly than young scid mice even though no immune reaction against these normal
stem cells could develop. This decrease in vitality would be due to factors that are not directly
related to the immune system such as an accumulation of mutations over time and damage to
the tissue environment. Therefore, old scid mice receiving young CD34+ cells are predicted
to heal more quickly than old CD34- scid mice. Likewise, if the proposed hypothesis is
correct, then the CD34+ scid old mice are expected to heal more quickly than the CD34+ wt
old mice. Although predicting relative healing rates between two groups with more than one
variable difference between them is difficult, a diagram illustrating one possible scenario of
results is illustrated in Figure 2.

Figure 2: Possible results for the recovery times of the different groups if the immune system
does inhibit stem cells in old age. Estimating the relative recovery times between groups with
more than one differing variable is difficult so this is just one possibility. If the hypothesis is
correct though, the CD34+ scid old mice should heal faster than the CD34+ wt old mice.

Confirmation of the expected results would support the hypothesis, but not prove it.
Other factors that are not related to the antigen recognition of stem cells, but are related to a
fully functioning immune system, could inhibit the healing process more in the wt mouse than
in the scid mouse. For example, perhaps a fully functioning immune system with B and T
cells could produce more inflammation at a wounding site and inhibit the healing process
even if those B and T cells do not recognize an antigen specifically. Although the results from
this experiment cannot absolutely prove the validity of the hypothesis, a negative outcome
would disprove the hypothesis. If the CD34+ scid mice heal insignificantly faster, the same
as, or slower than the CD34+ wt mice at both ages, the result would strongly suggest that the
adaptive immune system is not inhibiting the wound healing process with age. The results of
these experiments combined with specific assays for recognition of certain antigens by the
immune system should provide some very interesting data about the role the adaptive
immune system plays in the aging process.
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