Chapter 2

Serial Transplantation of Bone Marrow to Test Self-renewal

Capacity of Hematopoietic Stem Cells In Vivo
Charusheila Ramkumar, Rachel M. Gerstein, and Hong Zhang

Abstract
Hematopoietic stem cells (HSCs) have the ability to self-renew and replenish the blood and immune system
for the life span of an individual. An age-associated decline in HSC function is responsible for the decreased
immune function and increased incidence of myeloid diseases and anemia in the elderly. The changes in
HSC function are thought to occur as the result of an intrinsic defect in the self-renewal potential of HSCs
as they age. In this chapter, we describe a bone marrow serial transplantation protocol designed to test the
self-renewal capacity of HSCs in vivo.

Keywords Hematopoietic stem cells, Serial transplantation, Stem cell self-renewal, Stem cell aging,
Bone marrow

1 Introduction

Tissue-specific or adult stem cells are capable of self-renewal to

preserve stem cell pools and differentiation into a variety of effec-
tor cells. With advancing age, the self-renewal capacity of stem cells
invariably declines, eventually leading to the accumulation of unre-
paired, damaged tissues in old organisms (1). Hematopoietic stem
cells (HSCs), which give rise to various cellular components of
blood, are known to exhibit a decline in self-renewal capacity with
age. This is thought to contribute to decreased immune function
in the elderly. The age-associated changes in HSC function have
been extensively studied in mice, which include a strain-dependent
increase in HSC number (2–5) and a decrease in their lymphoid
differentiation potential (2, 4, 6). These changes are thought to
occur due to an intrinsic defect in HSC self-renewal potential as
they age (1).
Bone marrow transplantation is used to measure the “stem-
ness” of HSCs, as a single HSC is sufficient to reconstitute the
entire hematopoietic system of lethally irradiated recipients (7, 8).
The fundamental principle of stem cell self-renewal is established by

Kursad Turksen (ed.), Stem Cells and Aging: Methods and Protocols, Methods in Molecular Biology, vol. 976,
DOI 10.1007/978-1-62703-317-6_2, © Springer Science+Business Media, LLC 2013

17
18 Charusheila Ramkumar et al.

the ability of bone marrow from such recipients to reconstitute

secondary recipients with cells that are originated from the primary
transplanted cells (7, 9). As a gold standard test for long-term self-
renewal and multi-lineage potential of HSCs, serial transplantation
of bone marrow cells is able to reconstitute lethally irradiated recipi-
ents in successive but limited transplants, reflecting the finite poten-
tial of HSC self-renewal (10–12). It has been shown that serial
transplantation leads to a dose-dependent decrease in self-renewal
capacity of HSCs (13–15), and this decline in stem cell function
increases with the number of transplantations (10, 11, 16–19).
Stem cell exhaustion has been reported in serial transfer experiments
when unfractionated bone marrow (12, 18) or purified HSCs (20)
are transplanted. The stem cell exhaustion in serial transplantation
has been likened to an accelerated aging process, thus making it a
powerful system to study HSC aging. Here we describe a protocol
to test the long-term HSC (LT-HSC) self-renewal capacity using
serial transplantation of unfractionated bone marrow.

2 Materials

2.1 Mouse Strains 1. Donor mice: Mice with the genotype of interest on a C57BL/6
background. Age-matched wild-type C57BL/6 littermates are
used as controls.
2. Recipient mice: B6.SJL-Ptprca Pepcb/BoyJ (CD45.1+) aged
8–10 weeks (The Jackson Laboratory). This congenic strain
with an allelic variant CD45.1 antigen can be distinguished
from the C57BL/6 (CD45.2+) donor by flow cytometric
detection of the CD45 antigens. The use of CD45.1+ recipient
mice allows the determination of the contribution of the donor
cells (CD45.2+) to reconstitution of bone marrow in lethally
irradiated recipients, separate from residual recipient HSCs
(CD45.1+) that survive radiation and subsequently give rise to
hematopoietic cells marked by CD45.1.

2.2 Antibiotics 1. Neomycin (200×): 5 g dissolved in 50 ml distilled water, and

sterilized with a 0.45 μm filter.
2. Polymyxin-B (200×): 1 million units dissolved in 50 ml dis-
tilled water, and sterilized with a 0.45 μm filter.

2.3 For Irradiation 1. Cesium-137 radiation source.

2. Radiation chamber for mice.

2.4 For Harvesting 1. Harvesting medium: Biotin, flavin and phenol red-deficient
Bone Marrow RPMI-1640 medium (Invitrogen) supplemented with
10 mM HEPES (pH 7.2), 1 mM EDTA, and 2% fetal bovine
serum (FBS).
 Bone Marrow Serial Transplantation in Mice 19

2. Viability staining solution (1,000× stock): 3 mg/ml of acridine

orange and 5 mg/ml of ethidium bromide dissolved in dis-
tilled water. Prepare 100× solution with PBS.
3. 70% Ethanol.
4. Razor blades, dissecting scissors, and forceps.
5. 5 ml syringes with needles (25 G1/2 and 18 G).
6. 60-mm tissue culture plates and 15 ml Falcon tubes.
7. 70 μm nylon mesh (autoclaved).
8. Hemocytometer.
9. Temperature controlled centrifuge.
10. Fluorescence microscope.

2.5 For Injections 1. Isoflurane.

2. Sterile Dulbecco’s phosphate buffered saline (PBS).
3. Insulin syringe with fitted needle (29 G1/2).
4. Mouse restrainer.
5. Heat lamp.

2.6 For Flow 1. Antibodies: Lineage cocktail contains biotin-conjugated Ter119

Cytometry Analysis (clone TER-119), CD11b (clone M1/70), Ly-6G (Gr1, clone
RB6-8C5), CD45R (B220, clone RA3-6B2), CD19 (clone
1D3), and CD3e (clone 145-2C11). Additional antibodies for
HSC analysis include Ly-6A/E (Sca1)-FITC (clone D7),
CD117 (c-Kit)-PE-Cy7 (clone 2B8), CD135 (Flt3)-PE (clone
A2F10), and CD150-APC (clone mShad150). Other materials
include CD45.1-APC-eFluor 780 (clone A20), CD45.2-Alexa
Fluor 700 (clone 104), streptavidin-eFluor 450, and Fc block
CD16/CD32 (clone 2.4G2, from BioXCell). All except Fc
block are purchased from eBioscience.
2. Staining medium: Biotin, flavin and phenol red-deficient RPMI-
1640 medium (Invitrogen) supplemented with 10 mM HEPES
(pH 7.2), 1 mM EDTA, 2% FBS, and 0.02% sodium azide.
3. 96-well flexible plates and 5 ml polystyrene tubes.
4. LSR II flow cytometry system with 5 lasers and 18 detectors
(BD Biosciences).

3 Methods

3.1 Antibiotic Recipient mice are treated with antibiotics in drinking water 24 h
Treatment prior to exposure to radiation. Add 2 ml each of 200× antibiotic
stock solutions to 396 ml of autoclaved acidified water. Drinking
water with antibiotics must be changed twice weekly until 1 month
after transplantation (see Note 1).
20 Charusheila Ramkumar et al.

3.2 Irradiation Recipient mice are exposed to a lethal dose of 10 Gy (1,000 Rads)
whole body radiation using a Cesium-137 source (see Note 2). At least
five recipient mice are needed for each donor. For later (>3) cycles of
transplantation, at least ten recipient mice are used per donor.

3.3 Harvesting Donor Harvest bone marrow on ice in a laminar flow hood. Using sterile
Bone Marrow techniques is essential while flushing and preparing bone marrow
for injection.
1. Euthanize the donor mouse with isoflurane and cervical dislo-
cation, and immerse the mouse in 70% Ethanol completely.
2. Cut the hind limbs away from the hip joint. Be careful not to
break the femur while dissecting the hip joint. Similarly, cut
the forelimbs away from the shoulder joint. Place dissected
limbs in a 60-mm plate with cold bone marrow harvesting
medium on ice.
3. Hold one dissected limb with a pair of forceps, and scrape away
skin and muscle with a razor blade until only bone remains.
Try to get rid of as much tissue that is attached to the bone as
possible. Repeat this procedure with all limbs.
4. In a separate 60-mm plate with cold bone marrow harvesting
medium on ice, disarticulate the knee joint by cutting through
it with a razor blade. Cut tibia, femur, and humerus bones at
both ends so that marrow cavities are open.
5. Fill a 5 ml syringe with bone marrow harvesting medium and
fit a 25 G1/2 needle on the syringe. Hold the bone with a pair
of forceps. Fit the needle into one of the cut ends of the bone
and flush the bone marrow out. Repeat flushing until the color
of the bone changes from a pinkish tinge to almost completely
white. Repeat this procedure with all bones.
6. When all marrow has been flushed out, change the 25 G1/2
needle to an 18 G needle. The marrow is in large chunks and
can be broken up into a single cell suspension by passing it
through an 18 G needle several times.
7. Once a single cell suspension has been achieved, filter these
cells through a 70 μm nylon mesh into a 15 ml Falcon tube.
Bring the final volume of cells to 10 ml with staining medium.
Place cells on ice.

3.4 Cell Counting 1. Mix 10 μl of cell suspension, 89 μl of staining medium, and

1 μl of 100× viability stain solution by pipetting. Add 10 μl of
this mixture in the hemocytometer.
2. Count green (viable) cells in a 5 × 5 grid under fluorescence
microscope. Red/orange cells are dead (ethidium bromide-
positive) and can be counted in order to determine the ratio of
live to dead cells in a sample.
 Bone Marrow Serial Transplantation in Mice 21

3. Calculate concentration and total number of live cells:

Cells / ml in hemocytometer
= # green cells in the 5 ´ 5 square ´ 10 4
Cells / ml in tube = cells / ml in hemocytometer ´ 10
Total live cells in tube = cells / ml in tube ´ 10

3.5 Preparation 1. 2 × 106 bone marrow cells (~200 HSCs) per recipient mouse
of Bone Marrow Cells are usually used. Calculate the volume of cells sufficient for
for Injection required injections plus two extra injections (see Note 3).
2. Spin down cells at 1,500 rpm (388 × g) for 5 min in a centri-
fuge prechilled to 4°C. Resuspend cell pellet in 10 ml sterile
Dulbecco’s PBS.
3. Spin down cells again and resuspend cells in Dulbecco’s PBS at
a concentration of 2 × 106 cells per 200 μl. Place resuspended
cells on ice and bring them to mouse facility for injection.
4. The remaining cells are centrifuged and resuspended in stain-
ing medium at 6 × 107 cells/ml. They are used to stain for
the LT-HSC population in flow cytometry analysis (see
Subheading 3.7).

3.6 Bone Marrow Tail vein or retro-orbital injection can be used to inject donor bone
Cells Injection marrow cells into recipient mice. Tail vein injection is commonly
used, but it can be difficult to visualize tail veins in C57BL/6 mice.
Alternatively, cells are injected into the retro-orbital sinus of the
mouse in retro-orbital injection. While this method is technically
less challenging, it has the limitation of being able to inject a maxi-
mum volume of 200 μl into the sinus. In addition, if the needle
scrapes the cornea while injecting, the chances of developing cor-
neal ulcers are high. Both methods of injection require practice,
and we recommend practicing injection with PBS a few days before
the actual experiment.

3.6.1 Tail Vein Injection 1. Prepare the sample for injection by filling 200 μl of cell suspen-
sion into an insulin syringe and keep aside (see Note 4).
2. Place an irradiated recipient mouse in a mouse restrainer.
Warm the tail by shining a heat lamp on the tail briefly. This
causes vasodilation and enables easy visualization of the tail
veins.
3. Hold the tail in one hand, and select the vein you want to
inject. There are two veins, lateral and medial, in each tail.
Insert the tip of the needle into the vein, and withdraw slightly.
If blood is drawn into the syringe, the needle is in the vein.
Inject the cells quickly (see Note 5).
22 Charusheila Ramkumar et al.

3.6.2 Retro-orbital 1. Anesthetize the recipient mouse with isoflurane as follows.

Injection Soak a nestlet with isoflurane and place it in an empty cage.
Place the mouse in this cage and wait until its respiration slows.
This usually takes about 30 s.
2. Once respiration has slowed, take the mouse out and open an
eye wide by spreading the lids with one hand. The retro-orbital
sinus will be visible as a small opening at the medial corner of
the eye.
3. Place the tip of the needle in the sinus, hold the syringe at an angle
of 45° to the eye and inject cells into the sinus (see Note 6).
4. Place the animal back in its cage and observe until it awakens
from the anesthesia completely.

3.7 Fluorescence- 1. Cells resuspended at 6 × 107/ml in staining medium are incu-

Activated Cell Sorting bated with anti-CD16/CD32 antibody at 1 μg/106 cells for
Analysis of LT-HSCs 10 min on ice to block the Fc receptors.
in Bone Marrow 2. 25 μl of these cells are then incubated with 25 μl of each pri-
mary antibody in staining medium for 20 min in a 96-well
flexible plate on ice.
3. Spin down cells at 1,500 rpm (388 × g) for 5 min in a centri-
fuge prechilled to 4°C, and resuspend cell pellets in 100 μl of
staining medium. Repeat the washing step two more times.
4. Cells stained with biotin-labeled antibodies (lineage cocktail)
are incubated with streptavidin-eFluor 450 for 15 min on ice
and washed three times with staining medium.
5. After the final wash, cells are resuspended in 1 μg/ml propidium
iodide (PI) in staining medium for the exclusion of dead cells.
6. Flow cytometry analysis is performed on a 5-laser, 18-detector
LSR II fluorescence-activated cell sorting (FACS) machine
using 405, 488, 561, and 633-nm lasers. Data are analyzed
using FlowJo software (Treestar). A representative experi-
ment in Fig. 1 shows the progressive gating strategy used to
analyze LT-HSCs from live bone marrow cells (PI-negative).
Lineage negative (Lin−) cells are those lacking significant
expression of Gr-1, CD11b, Ter119, CD3, B220, and CD19.
Long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs),
and multipotent progenitors (MPP) are characterized by
Lin−Sca1+c-kit++CD150+Flt3−, Lin−Sca1+c-kit++CD150−Flt3−,
and Lin−Sca1+c-kit++CD150−Flt3+, respectively (21, 22).

3.8 Monitoring Mice 1. After transplantation, recipient mice must be monitored daily
and Subsequent for signs of ill-health including pallor, ruffled fur and lethargy.
Transplantations Typically, recipient mice receiving inadequate injections will
become increasingly pale and sick, and die between 10 days
and 2 weeks after irradiation due to bone marrow failure.
 Bone Marrow Serial Transplantation in Mice 23

Fig. 1 A representative FACS analysis of LT-HSCs in bone marrow of a wild-type C57BL/6 mouse. Gates used
for FACS analysis are displayed as blue boxes. The frequency of each gated population as a percent of the
displayed cells is shown in red. Only live cells (propidium iodide excluding) are displayed. Lineage negative
(Lin−) cells (left panel ) are gated and then displayed in the middle panel. LSK (Lin−Sca1+c-Kit++, middle panel)
cells are gated and displayed in the right panel

2. When 2 months after the injection have elapsed, the stem cell
numbers reach homeostasis and secondary transplants can be
performed. These recipients can be used as donors for the next
transplantation. With every ensuing transplant, a sequential
decrease in the frequency of the LT-HSC population is
expected. HSCs from wild-type mice normally can reconstitute
recipient bone marrow for 4–5 cycles of transplantation before
stem cell exhaustion occurs.
3. Relative contributions of the donor and residual recipient
bone marrow to the reconstitution in recipient mice can be
determined by staining for CD45.1 and CD45.2 of bone mar-
row from the first transplantation onward. More than 90% of
the cells are usually derived from the donor (CD45.2) bone
marrow.

4 Notes

1. Leave a note on cages of recipient mice that they are on antibi-

otic treatment, so animal care technicians do not change the
water.
2. Set up irradiation late in the afternoon, and irradiated mice can
be transplanted with fresh bone marrow the next morning.
Make sure to check the Cesium-137 source to calculate the
radiation dosage every time you irradiate.
3. If injecting five mice, calculate the volume of cells required for
14 × 106 cells (5 plus 2 extra injections, 2 × 106 cells/injection).
4. Before injection, make sure that the cells are not chilled by
warming them between your hands briefly.
5. Watch for clearing of the vein lumen; this indicates a successful
injection. If you feel any resistance while injecting, it means the
24 Charusheila Ramkumar et al.

needle has slipped out of the vein. If that happens, withdraw

the syringe and reinsert it into the vein proximal to the original
injection site.
6. If cells regurgitate back, that means they are not in the sinus.
Take out the needle and reinsert. If cells come out through the
nose, the procedure is unsuccessful and must be repeated. This
process has to be performed quickly, as the effect of isoflurane
wears off within 1–2 min.

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