Banking Your Baby's Cord Blood

A serious option for pregnant parents to consider

by Robert Sears, M.D.
When my wife was pregnant with our third child, I remember sitting in our
obstetrician's (OB) waiting room together while my wife flipped through the latest
pregnancy magazine.
"Hey honey, look at this!" I heard my wife say. She showed me an article about
umbilical cord blood stem cell banking and asked me if we should look into it.
I discovered it was much simpler than I'd imagined. After the umbilical cord is cut, the
blood is drained out of the placenta and remaining umbilical cord, thus the term,
"cord blood." This blood is rich in baby's "stem cells," which are immature blood cells
that are able to change and mature into any type of blood cell as baby grows, just
like bone marrow cells. Today stem cells are being used to treat over seventy-five
different cancers and blood and immune disorders.
My wife felt strongly about banking our baby's cord blood because she had cancer as a
teenager. She wanted to take every precaution for our kids, and having some cord
blood available in case any of them should need it gave her peace of mind. And, she
could even use it herself. Of course, we hoped we would never need it for these
reasons, but as I read more about stem cells, I found out there were many more uses
than just treating cancer. This is really what got me excited about banking our baby's
blood. I decided to invest in our family's future.
The benefits of family cord blood banking
Cord blood stem cells are not just for your baby. It's really an investment for the
whole family. And while the chance that any family member will use the cord blood
for cancer treatment is very low, the likelihood that it could be used to treat a variety
of diseases is considerable. Scientists have discovered significant potential for the use
of stem cells in treating conditions such as coronary artery disease, nerve and brain
damage, strokes, multiple sclerosis, and cardiovascular disease, the number one
cause of death in this country. If stem cell treatments become a viable and routine
option for preventing and treating cardiovascular disease, then having banked stem
cells will be an enormous advantage.
Cancer and other blood-related disorders
Besides these exciting possibilities, there are still the current uses for treating certain
cancers and other blood disorders. Here are some benefits when cord blood is used
instead of bone marrow:

Research shows survival rates more than double when a person's own cord blood or a
family member's cord blood is used, compared to using an unrelated donor sample
from a public stem cell bank (63 percent survival with related cord blood, 29 percent
survival with unrelated cord blood). Plus, having your own sample stored ensures
immediate availability and minimizes the risk of waiting for a donor.

Cord blood stem cells don't need to "match" the recipient as perfectly as bone
marrow, thus increasing the chance that a family member can receive a related stem
cell transplant.

Patients who receive cord blood stem cell transplants have a smaller chance of
rejecting the cells, compared to bone marrow stem cell transplants.

During my pediatric training I spent two months in the Children's Hospital Bone
Marrow Transplant ward. I watched numerous kids undergo these transplants. Kids
who used their own stem cells, or a family member's cells, fared much better. It
provides some peace of mind that if ever our family is faced with such a challenge,
we will have better treatment options available to us.
How cord blood is collected and stored
Before your due date, the cord blood bank sends you a collection kit that contains
everything your doctor needs for the collection. When your baby is born and the
umbilical cord is cut, your OB or midwife collects the blood from the remaining
umbilical cord and placenta into a syringe or blood bag. The process only takes a few
minutes, and can even be collected during a C-section.
A family member places the cord blood into the preaddressed mailing package and
makes one phone call to a medical courier. Within hours the cord blood is picked up
and shipped overnight to the cord blood bank. Once there, it is processed. The stem
cells are removed from the cord blood, and are then placed into liquid nitrogen for
storage. Choosing a cord blood bank
Deciding WHOM we should trust to do the banking was a challenge. There are several
companies to choose from, and I spent days reading their literature and scrutinizing
their Web sites before we chose Cord Blood Registry.SM See the box below to learn
more about our decision.
Deciding whether to bank your baby's cord blood is a personal decision and a financial
commitment. But parents only have one chance with each child to take advantage of
this technology. When choosing where to store your child's cord blood cells, it's
important to ask questions and research your decision carefully. For more
information, go to www.cordblood.com.
Robert Sears, M.D., is a nationally recognized pediatrician and author who is in
private practice in Southern California.

Here are some reasons why we chose

Cord Blood RegistrySM:

CBR has facilitated more

transplants than any other family bank.
It has provided more than thirty stored
units for transplants. This was
important to me because I felt if a
bank has never used a single sample,
how do they know the samples are
viable and being stored properly?

CBR was the first cord blood

bank to become accredited by the
AABB and is a proven leader in the
industry.

CBR currently has processed

over 260,000 units of cord blood and
owns and operates its own storage
facility.

CBR stores its samples in

multiple vials. Scientists are currently
working on techniques to expand the
stem cells stored in a single cord blood
sample. When the technology becomes
available, it will be advantageous to
have your sample stored in multiple
cryovials.

CBR is a financially strong

company whose lab has been
processing and storing cord blood since
1992. This is crucial because you want
your samples to still be around in
twenty years or more.

Frequently Asked Questions

Cord Blood Banking
Cord blood banking is an opportunity for
parents to save the excess supply of
newborn stem cells from their babies'
umbilical cord blood as a type of biological
investment because the genetically unique
stem cells have current and potential uses
in medical treatment. Expecting parents
have many questions about the opportunity
to save stem cells at birth. The following
are the most common questions asked
about cord blood banking:
1. What are stem cells?
Stem cells are the "master cells" that
produce all the various cell types in the
body. The stem cells that are primarily used
in medical therapies can be found in the
blood that circulates through your body, in
bone marrow, and in umbilical cord blood
(the blood that remains in the umbilical
cord after delivery). Stem cells are a
lifesaving resource. They are not only used
to treat a variety of cancers, blood
disorders, and genetic diseases, but
researchers believe they have great

potential to help treat a variety of

common conditions such as diabetes,
heart disease, and stroke.
2. Are newborn stem cells different
than other stem cells?
In many ways, cord blood is an
ideal source for stem cells. Unlike
embryonic stem cells derived from
fetal tissue, there are no moral
questions or controversies regarding
newborn stem cells. In fact, many
researchers are turning to newborn
stem cells to avoid the moral conflicts
of embryonic cells. Additionally, unlike
embryonic stem cells, newborn stem
cells already have established and
proven treatment protocols for use in
humans. When compared to adult stem
cells, newborn stem cells are
advantageous due to their higher rates
of
proliferation1;
immunological
naivete2; and reduced exposure to
viruses and aging.
3. What is cord blood banking?
Cord blood banking is an
opportunity at birth that enables
expectant parents to have their
newborns' cord blood collected and
cryopreserved for future use. When
you save cord blood for your baby, the
cells are specifically reserved for your
family.
4. What diseases are treated with stem
cell transplantation?
Currently, newborn stem cells
are used to treat many of the same
diseases associated with bone marrow
stem cell transplants, including certain
blood disorders, cancers, and genetic
diseases. In total, there are over 75
diseases that may be treated with
newborn stem cells. In addition,
researchers worldwide have been

focusing on the potential of newborn stem

cells to treat common conditions such as
heart disease and diabetes. Recent
advancements in the experimental use of
newborn stem cells to treat spinal cord
injuries and brain injury have been
groundbreaking. The mounting evidence of
the ability of newborn stem cells to treat a
wide variety of additional diseases is very
compelling.
5. Is cord blood collection safe?
Absolutely. The blood in a baby's
umbilical cord is normally discarded with
the cord after it is clamped and cut. When
you ask for your baby's cord blood to be
collected, the one and only change from
the normal procedure is that after birthafter the cord is cut-the baby's cord blood is
collected rather than thrown away.
Collections can take place even after the
placenta has been delivered.
6. Who can use my newborn's stem cells?
Your baby's stem cells are, of course,
a perfect genetic match for your baby;
however, there are certain diseases that
cannot be treated with one's own stem
cells. In those cases, doctors would first
look to a sibling as a donor source. The
baby's stem cells may be a suitable match
for the mother due to her carrying the child
during pregnancy.3 The cells may be useful
for the baby's siblings, father, or cousins if
there is an adequate HLA match.
7. How much does it cost to save newborn
stem cells?
Cord blood banking is a highly
specialized field requiring precise cell
processing and testing. Costs for cord blood
banking vary depending on the quality and
experience of the bank. Accredited cord
blood banks generally charge between
$1,500 and $2,000 for first-year costs and
about $125 annually for storage.

8. Why do families save newborn stem

cells?
Newborn
stem
cells
are
considered one of the most promising
medical treatments of the future. Most
families bank their babies' cord blood
for peace of mind, knowing that the
current and future uses for stem cells
may be valuable to their family.
Banking cord blood allows immediate
access to related stem cells for
someone in your family who may need
a transplant. This is important because
studies
have shown
stem
cell
transplants from a relative have
resulted in double the survival rates
compared to transplants with stem
cells from unrelated donors.4
9. Should I save cord blood for all of
my children?
Each child is genetically unique.
Saving cord blood from all children in a
family provides related sources of stem
cells for potential use and increases
the likelihood of a useful match. The
stem cells in a sibling's cord blood are
up to twice as likely to be useful for
transplant compared to stem cells from
the same sibling's bone marrow. In the
case of identical twins, it is still
important to save as many stem cells
as possible, and it is recommended
that cord blood be collected for both
babies. In general, the collection
volume per baby in multiple births is
smaller, so collecting for both babies
helps ensure an adequate stem cell
yield for transplantation if ever
needed.
10. What are the odds that my family
will need to use the newborn stem
cells?

According to published data, the

odds that a child will someday need to use
his or her own newborn stem cells for
current treatments are estimated at 1 in
400.5 Odds that the newborn or a family
member may benefit from banked cord
blood are estimated at 1 in 200.5 These
odds do not include the potential use of
newborn stem cells to treat heart disease,
diabetes, Parkinson's, Alzheimer's, and
spinal cord injury. The continued progress in
medical treatments would greatly increase
the likelihood of use by your baby
throughout her life. Based on current data,
there is no "expiration date" for newborn
stem cells.
11. How long can the cells last in storage?
Stem cells should remain viable indefinitely
when stored properly. Stem cells frozen for
15 years proved viable for use in
transplantation.6 The New York State Health
Department Guidelines for cord blood
banking claim: "There is no evidence at
present that cells stored at -196C in an
undisturbed manner lose either in-vitro
determined viability or biologic activity.
Therefore, at the current time, no
expiration date need be assigned to cord
blood stored continuously under liquid
nitrogen."
12. What other options do I have regarding
my baby's cord blood?
While you are making your decision about
family banking, you have the opportunity to
enroll in a public service program that will
help you save cord blood for your newborn's
own potential use if he or she scores 6 or
less on the 1O-minute Apgar test following
birth. Signing up for the Newborn
possibilities ProgramSM is a step towards
giving your newborn access to his or her
own stem cells, which may potentially
provide medical advantages to your baby.
Another option for some families is the

Designated Transplant Program, which

provides cord blood banking for
qualified families with an acute
medical need for stem cells. Learn
more about the Newborn Possibilities
Program and the Designated Transplant
Program
online
at
www.cordblood.com/care.
Another option for you might be public
donation, but only if you are delivering
in a hospital that has a donation
program. Check with your hospital and
also
learn
about
eligibility
requirements for donation. Many
samples may not be collected or saved
based on family medical history,
maternal medical history, or collection
volume. It is important to recognize
that there is no guarantee that your
baby's own stem cells will be collected,
saved, or available to your family in
the future if you participate in a public
donation program.
13. If someone in my family needs a
transplant, couldn't we find a donated
sample from a public bank?
Hopefully.
However,
many
patients are unable to locate a donor,
especially those who belong to ethnic
groups that are not adequately
represented in the public banks. Stem
cells from a relative (preferably a
sibling) are generally the best
treatment
option
in
transplant
situations. In fact, one study has shown
the one-year survival rate for patients
treated with a sibling's newborn stem
cells is approximately 63%. The survival
rate with stem cells from unrelated
donors drops to only 29%.4 Related
newborn stem cells also reduce the risk
of graft vs. host disease (GVHD), a
leading complication of stem cell
transplants. More importantly, because

the immune cells are immature, cord blood

is twice as likely to be a suitable match
between siblings when compared to bone
marrow. Public banks may be able to help
provide an adequate sample for transplant,
but it may not be the best or most
preferable sample.
Is cord blood banking worthwhile?
Only you can decide what is right for your
family. Some families feel that cord blood
banking is too speculative or cost
prohibitive, while others see it as a nominal
cost for the peace of mind and future
potential benefits that may be realized.
Newborn stem cells may literally be
lifesaving to your child or family member.
Conversely, there is no guarantee that the
cells will ever be used.
REFERENCES
1 Cairo. MS and Wagner, JE. Blood. 1997;
90(12):4665-4678.
2 Stephenson, J. Journal of the American
Medical Association. 1995; 273:1813-1815.
3 Harris D, Schumacher M, LoCascio], et
al. Bone Marrow Transplantation.
1994;14:63-68.
4 Gluckman. E. et al. New England
Journal of Medicine. 1997; 337 (6):373-381.
5 Pasquini MC, Logan BR, Verter F, et al.
The Likelihood of Hematopoietic Stem Cell
Transplantation (HCT) in the United
States: Implications for Umbilical Cord
Blood Storage. Blood. 2005;106(11).
6 Broxmeyer H, Srour E, Hangoc G, et al.
Proceedings of the National Academy of
Sciences. 2003;100(2):645-650.
In addition to the current uses, new
medical applications for newborn stem cells
are being discovered rapidly; however,
banking cord blood does not guarantee that
the cells will provide a cure or be
applicable for every situation.

This educational information was made

possible through funding from Cbr
Systems, Inc.-Cord Blood Registry.
Information is available online at
www.cordblood.com or by calling 1888-CORD BLOOD (1-888-267-3256).

Cbr Systems, Inc. 2006.

0906 MAOO747.03

True Transplant Story

Cord Blood Saved Our Sons Life
Joseph and Darlene Davis of Cedar Hill,
Texas, are the proud parents of two
healthy sons, three-year-old Joseph Jr.
and one-year-old Isaac; the baby
Darlene calls her miracle child.
Joseph Jr. wasn't always the
strong and healthy boy he is today.
Diagnosed at birth with sickle
cell anemia, Joseph Jr. lived in
constant pain. He ran high fevers,
suffered from . fal1l\\')1 swollen 1ne
DaVIS hands and feet and required
frequent blood transfusions to control

his red blood cell count. To run and play

like other kids his age, Joseph Jr. would
need a stem cell transplant. 'The doctors
told us that our son may not be with us for
very long," said Joseph Sr. "We didn't even
know if he would make it to his teens."
Joseph )r.'s parents were determined to
help their son. They spent a year searching
for a stem cell donor. "Being African
American made it so much more difficult,"
added Darlene.
The discouraged parents worried constantly
about their son, until a lucky twist of fate
turned the family's life around.
Darlene was pregnant. At first the couple
couldn't believe it. They had undergone
infertility treatments to get pregnant with
Joseph Jr. and never counted on having
another child. "I was told I couldn't get
pregnant again," said Darlene.
recalls the moment the doctor told her the
news, "He said, 'this is a miracle,' and I said
'yes it isl'" Darlene gave birth to a healthy
baby boy. "I was happy, so I named my baby
Isaac because that means laughter," said
Darlene.
Minutes after Isaac was born, doctors
collected the blood from his umbilical cord.
His stem cells were banked with Cord Blood
Registry, the world's most experienced
family cord blood bank, free of charge as
part of its Designated Transplant Program~
-a service provided to families based on
medical need.
Prior to the transplant, Joseph Jr. received
high doses of chemotherapy that destroyed
his blood system, which was creating the
sickle cells. His physician, Dr. Joel
Weinthal, pediatric hematologist and stem
cell transplant physician explained, "once
we wiped out his old system with high-dose
chemotherapy, the new donor cells from
the cord blood could be given to form an
entirely new blood and That Isaac was there

to save immune system, which Joseph,

that's the miracle. woul~ ~,ot have
sickle cell anemIa.
Isaac's cord blood stem cells were
transplanted into his brother on May
10, 2002. "It was about twenty days
after the transplant, when all of a
sudden he started feeling better," said
Joseph Sr.
"He started looking good, his energy
was back, and he was on his way to
recovery."
The couple was elated. Amniocentesis
revealed that the baby Darlene was
carrying was a perfect match for
Joseph Jr. She
I
It's been a little over a year since
Joseph Jr.'s cord blood transplant. Now,
he is a healthy, vibrant three-year-old.
His doctors say he is cured. At home,
the boys play and wrestle and
according to their parents, are as close
as brothers can be. "That Isaac was
there to save Joseph, that's the
miracle," said Joseph Sr. "He doesn't
really understand about cord blood
now, but he knows something about
Isaac helped save his life."

Half of All Americans Could Benefit

from Stem Cell Research
Experts are predicting that stem cell
research has the potential to help up
to half of all Americans, who suffer
from some form of presently incurable
disease, injury or birth defect. Some
of those conditions include:
one million children with juvenile
diabetes 8.2 million people with cancer
58 million with heart disease four
million suffering from Alzheimer's
disease 10 million with osteoporosis 43
million arthritis sufferers 250,000
people paralyzed by spinal cord
injuries 30,000 victims of Lou Gehrig's
disease.
For some Americans, however, living
proof of the healing power of adult
stem cells has long been evident. Early
successes using adult stem cells have
turned scientific theory into

groundbreaking treatments and has

offered victims a second chance at
a normal life. The progress being
made using embryonic stem cells and
their potential for greater
versatility has added considerably
to the expectations of fmding cures
for otherwise incurable diseases in
the near future. "In fact, stem
cell research is such a young
field," says Eve Herold, of the
Stem Cell Research Foundation, "we
will probably discover other uses
for these cells as the science
evolves. It's impossible to say, at
this stage of the game, what the
limitations will be." One stem cell
success story is that of Savannah
Jantsch, a 9-year-old girl from
Bellingham, Washington struggling
with leukemia, a blood cancer. On
November 26, 1997, she was treated
at the Fred Hutchinson Cancer
Research Center with blood stem
cells obtained from the umbilical
cord blood of a newborn. These stem
cells produced the blood cells she
needed to fight her cancer.
When Jesse Farquharson was four
months old, he was diagnosed with a
rare form of cancerous tumor in his
left eye, a condition called
bilateral retinoblastoma. Not long
after the doctors decided to remove
his left eye, they found that the
cancer had spread to his spinal
cord. On April 19, 2001, J esse
underwent a bone marrow transplant
using stem cells from his own
umbilical cord blood at the Toronto
Hospital for Sick Children. Jesse's
stem cells grafted sooner than
doctors had anticipated and he had
no complications. Today, Jesse's
parents credit stem cells with
saving their son's life. He remains
one of the few people in the world
to have undergone this type of
transplant.
Keone Penn made medical history
when, in December of 1998, he was
cured of sickle-cell anemia, a
painful genetic blood disease, in a
procedure that destroyed his
diseased blood through radiation
and then injected him with stem
cells.

Keone's condition was diagnosed when he

was 4 months old and was further
complicated by a stroke at the age of
5, which left him with an estimated
five years to live. A year after the
stem cell transplant, doctors found no
traces of the disease that had caused
him such intense pain and had stunted
his growth. Amazingly, the blood stem
cells he was infused with completely
replaced all the blood cells in his
body, even changing his blood type to
that of the stem cells. Now he believes
he can fulfill his dream of becoming a
chef and join in on basketball games,
activities that before now he had only
longed to do.
For more information about stem cell
research, visit the Stem Cell Research
Foundation's website at
www;StemCellResearchFoundation.org or
call 1-877-842-3442.
Stem Cell Research Foundation. 877-8423442 .
www.StemCeIIResearchFoundation.org

Blood from umbilical cord offers

medical benefits
By Brooke Adams Excerpt from The Salt
Lake Tribune
Merlaine and Ryan Waldron consider the
tiny vials of blood tucked away in a
cryogenic vault in Arizona as the best
investment they have made yet for their
two children.
The vials contain umbilical cord blood
collected at each child's birth Lucas, now 3, and Katherine, 3 months.
With a family history of adult-onset
diabetes, the Waldrons were willing to
take the "what-if' gamble that their
children eventually might need the
stem-ceIl-rich blood - though use of
stem cells to treat diabetes is merely
an area of research today.
"I would hate to look back 10 years
from now and say too bad we didn't
store your cord blood," said Merlaine

Waldron, who lives in West Valley

City. "I am glad we took advantage
of it." Once routinely tossed in
the trash, umbilical cords are now
becoming a hedge in the treatment
of such diseases as sickle cell
anemia, leukemia and other cancers.
Cord blood can be an expensive
investment and one critic says it
is unwarranted except for families
with proven histories of certain
diseases.
But that isn't stopping thousands
of new parents from signing up to
have their newborn's cord blood
stored at the dozen or so private
cord blood banks operating around
the country. Some parents are even
conceiving babies with the intent
of using cord blood stem cells to
aid an already ill sibling.
Others are donating the blood to
one of the nearly two dozen public
banks, such as those associated
with the American Red Cross or the
National Marrow Donor Program, that
make supplies available to anyone
in need.
The fluid is gathered within
moments of birth by a nurse or
doctor, using either a syringe or
blood bag to drain the placenta and
cord after it is cut. Once
delivered to a bank, it is
processed and stored in a cryogenic
vault. The most recent studies show
the fluid is viable for 15 years and perhaps longer.
Researchers and blood industry
experts figured out in the late
1980s that stem cells in cord blood
could be used the same way as those
gathered from bone marrow - with
added benefits.
Among the benefits: Collecting the
blood is painless and less risky
than a bone marrow transplant.
Because of their immaturity, cord
blood stem cells are less likely to
be contaminated with disease. The
cells are more likely to be a good
match for siblings, parents and
other relatives, are immediately
available and improve survival
rates. The cells also are easier to
match to unrelated individuals than
bone marrow.

Compared to bone marrow stem cells,

cord blood cells are less likely to
trigger an adverse reaction,
particularly in relatives.
Currently, stem cells are used to
regenerate blood and the immune system,
typically after chemotherapy or
radiation treatment for cancer. At
least one clinical study is using the
cells to grow heart tissue, and
researchers are looking at their use in
growing bone, nerve, skin and brain
tissue.
"A l~t of families are doing it not
just for what it can be used for now,
but what it will be used for in the
future," said Rita Kennen, spokeswoman
for Cord Blood Registry in San Bruno,
Calif., the nation's first and one of
the largest private banks.
It's that iffy future that concerns
some doctors. They say parents without
family histories of certain diseases
are making an emotional, rather than
scientifically justified, decision in
opting to store cord
Moments after an umbilical cord is cut,
the blood is gathered by a nurse or
doctor. A syringe or blood bag is used
to drain the placenta and cord. (Photo
courtesy of Cord Blood Registry)
blood in a private bank. That's
primarily because of the rarity of
contracting one of the diseases that
stem cells can treat - between l-in2,000 and l-in-3,000, according to a
Salt Lake City physician.
"It's probably not cost-effective,"
said Steven Clark, medical director of
the Maternal and Fetal Medicine Program
at LDS Hospital. "They are using
emotional pressure to get people to
sign up for something that may not be
beneficial." Collection fees charged by
private banks range from $400 to
$1,500, with annual storage fees of $50
to $120. Some banks have assistance
programs for families with a proven
medical need.
Public banks don't charge but also
don't guarantee your supply will still
be available should you need it.
Helen Ng, spokeswoman for the National
Marrow Donor Program, seconds Clark's
opinion.

Public banking, however, is a "good

thing to do because there is
someone out there in need," Ng
said. "It offers those without a
[bone marrow donor match] to have
another option. When a baby is
born, a mother is giving life
twice." There is a particularly
pressing need for donations from
minority families, who are
underrepresented in donor
registries and whose ethnic makeup
makes finding a match difficult. .
According to the National Marrow
Donor Program, there were 2,000
transplants using cord blood
through December 2000. Cord Blood
Registry's reserves have been used
in 28 transplants, most on behalf
of a sibling with some form of
leukemia.
.. . Kennen said her company
supports greater public supplies of
cord blood. But the availability of
a secure, genetically related
supply of stem cells is reassuring
to many families.
"For what it costs, you would spend
that amount on a widescreen TV you
would throwaway in three years,"
said Kennen, whose company began
commercial storage in 1995 and now
keeps 50,000 samples at its
facility in Tucson, Ariz.
Jayna and Tyler Haws, of Provo, are
among the firm's Utah clients who
like the added "insurance" of
providing for any future
eventualities. Jayna's parents, who
own an insurance agency in Boise,
Idaho, provided the coverage as a
gift to their granddaughter.
"It's cheap insurance for cancer or
other horrible things," Jayna Haws
said.
1103 MA00514.01

The Likelihood of Hematopoietic Stem

Cell Transplantation (HCT) in the
United States: Implications for
Umbilical Cord Blood Storage
Pasquini Me, Logan BR, Verter P,
Horowitz MM, Nietfeld n. Blood.
2005;106(1l):386a.
Poster presented at: 47th Annual
Meeting of the American Society of
Hematology (ASH); December 10-13,2005;
Atlanta, Georgia.
HCT effectively treats a variety of
malignant diseases. Availability of
suitable grafts limits application in
some cases, prompting the establishment
of banks to store umbilical cord blood
for later personal or family use. The
likelihood of utilizing stored cells
depends, in part, on the likelihood of
developing a condition for which HCT is
indicated. This study estimates the
latter likelihood based on data for
current HCT use as reported to the
CIBMTR from 2001 through 2003. First,
age-related incidences of HCT were
calculated; then, using the cumulative
incidence function, we calculated the
lifetime likelihood under two
scenarios. In scenario 1, we calculated
the likelihood of receiving an
autologous HCT by age, in decades, from
birth to 70 years. In scenario 2, we
calculated the likelihood of being a
candidate for either an autologous or
an allogeneic HCT. This likelihood was
derived from the number of HLAidentical sibling HCTs multiplied by
three (to account for patients
considered acceptable candidates but
lacking an HLA-matched sibling donor)
in addition to the number of autologous
HCT. The number of HCT performed

represented fewer than 20% of the

malignant diagnoses most commonly
treated with HCT, according to
Surveillance, Epidemiology and End
Results (SEER) data. After the
second decade of life, the agerelated incidences of HCT under
both scenarios steadily increased
with age. The cumulative incidences
for scenario 1 ranged from 0.02%
(at age 20) to 0.23% (at age 70);
for scenario 2 they ranged from
0.06% (at age 20) to 0.46% (at age
70) (figure below). Given the
current indications for HCT. the
lifetime likelihood of undergoing
an autologous HCT or being a
candidate for HCT is about 1 in 400
and 1 in 200. respectively. How
closely these estimates correspond
to the likelihood that a stored
cord blood unit is used depends
upon several conditions including
a) sufficient number of stem cells;
b) satisfactory stem cell quality
after the storage period; and, c)
relative attractiveness, in
particular clinical situations, of
cord blood cells compared to other
graft sources (e.g. peripheral
blood or bone marrow).
0.5%1 1-1
DScenario
~
0 0.3% 0
~
.~
QJ
~ 0.2%~ I
.~
0.1%~ 1 1

:200 . Scenario 1 O.4%i

2
High-efficiency recovery of functional
hematopoietic progenitor and stem cells
from human cord blood cryopreserved for
15 years
I I I . I 1-1 :500
1 I - 1 - 1 1-1:1000

10
20
30
40
50
60
70
Age Decade
Cbr Systems, Inc. . 0106

Hal E. Broxmeyer*H~, Edward F.

Srour*tll**, Giao Hangoc*~, Scott
Cooper*~, Stacie A. Andersontt, and
David M. Bodinett
Departments of *Microbiology and
Immunology, 'Medicine, and
IIPediatrics, Walther Oncology Center,
and **Wells Center for Pediatric
Research, Indiana University School of
Medicine, Indianapolis, IN 46202;
~Walther Cancer Institute,
Indianapolis, IN 46208; and ttGenetics
and Molecular Biology Branch, National
Human Genome Research Institute,

National Institutes of Health,

Bethesda, MD 20892
Communicated by Edward A. Boyse,
University of Arizona College of
Medicine, Tucson, AZ, November
20,2002 (received for review
September 7,2002)
Transplanted cord blood (CB)
hematopoietic stem cells (HSC) and
progenitor cells (HPC) can treat
malignant and nonmalignant
disorders. Because long-term
cryopreservation is critical for CB
banking and transplantation, we
assessed the efficiency of recovery
of viable HSC/HPC from individual
CBs stored frozen for 15 yr.
Average recoveries ( 1 SO) of
defrosted nucleated cells, colonyforming unitgranulocyte,
-macrophage (CFU-GM), burst-forming
unit-erythroid (BFU-E), and colonyforming unit-granulocyte,
-erythrocyte, -monocyte, and
-megakaryocyte (CFU-GEMM) were,
respectively, 83 12, 95 16, 84
25, and 85 25 using the same
culture conditions as for prefreeze
samples. Proliferative capacities
ofCFU-GM, BFU-E, and CFU-GEMM were
intact as colonies generated
respectively contained up to
22,500, 182,500, and 292,500 cells.
Self-renewal of CFU-GEMM was also
retained as replating efficiency of
single CFU-GEMM colonies into 2
dishes was >96% and yielded 2
colonies of CFU-GM, BFU-E, and CFUGEMM. Moreover, C034+C038- cells
isolated by FACS after thawing
yielded >250-fold ex vivo expansion
of HPC. To assess HSC capability,
defrosts from single collections
were bead-separated into C034+
cells and infused into sublethally
irradiated nonobese diabetic
(NOD)/severe combined
immunodeficient (SClO) mice. CD45+
human cell engraftment with
multilineage phenotypes was
detected in mice after 11-13 wk;
engrafting levels were comparable
to that reported with fresh CB.
Thus, immature human CB cells with
high proliferative, replating, ex

vivo expansion and mouse NOD/SCID

engrafting ability can be stored frozen
for> 15 yr, can be efficiently
retrieved, and most likely remain
effective for clinical transplantation.
Cord blood (CB) is a viable alternative
to bone marrow for related and
unrelated allogeneic hematopoietic stem
cell (HSC)jprogenitor cell (HPC)
transplantation (1-13). Since our
initial preclinical (14-16) and
clinical (1, 17-19) studies, there have
been >2,000 CB transplants performed to
treat a variety of malignant and
nonmalignant disorders in children and
adults (1-13, 17-19). CB HSCjHPC are
frozen before use for transplantation
(14, 20-22), but the longest that a CB
collection has been stored frozen
before use for clinical transplantation
is in the 3- to 5-yr range, With> 1
00,000 CBs stored frozen world-wide for
prolonged periods in anticipation of
their clinical use, information on
longer-term storage of CB HSC and HPC
is of critical importance.
The capacity to freeze and retrieve CB
HPC cells was first reported when we
suggested that CB could serve as a
source of transplantable and engrafting
HSC and HPC (14). We subsequently
evaluated effects of 5-yr (16) and 10yr (23) storage on retrieval of HPC in
which postfreeze HPC numbers were
compared directly to prefreeze numbers
from the exact same CB samples. Thus, a
true recovery rate could be calculated,
At those times, we assessed only
numbers and proliferation of HPC in
vitro. In the present report, we
extended analysis of HPC to 15-yr
cryopreservation, and evaluated HSC
content and en grafting capability of
defrosted cells as well as additional
important functional characteristics of
HPC. This information included the
www.pnas.orgjcgijdoijl0.l073jpnas.02370
86100
replating capacity of frozen CB
multipotential progenitor cells
[( colony-forming unit-granulocyte,
-erythrocyte, -monocyte, and
-megakaryocyte (CFU-G EM M) j, which
estimates the "selfrenewal" capacity of

these cells (24, 25), the ex vivo

expansion capability uf frozen CB
HPCjHSC, studies previuusly done on
cells stored frozen for very shurt
periods of time (26-28), and the
engrafting capability in vivo of
stored CB HSC in nonobese diabetic
(NOD)jsevere combined
immunodeficiency (SCID) mice. NOD
jSCID mice are currently considered
the best assay model for evaluating
human HSC (29-32) and may provide a
more direct assessment of the
anticipated behavior of thawed CB
cells in vivo. Our results
demonstrate that CB HSC and HPC can
be cryopreserved for at least 15
yr, and efficiently retrieved in
functionally competent form.
Materials and Methods
CB Cells: Collection,
Cryopreservation, and Separation.
CB cells used were scheduled for
discard after delivery of the
infant and prior needs, if any, for
samples for clinical study had been
satisfied.
The Institutional Review Buarduf
the Indiana University School of
Medicine approved this study. Cells
were frozen and subsequently
retrieved after thawing as reported
by us (14, 16,23). In short, CB
obtained within 36 h of collection
were separated by Ficoll-Hypaque
density cut procedure (density
1.070 g/cm3;
Pharmacia) into a low-density
1.070 gjcm3) mononuclear cell
fraction. The cells were washed,
and a small aliquot was used for in
vitro colony assay. The remaining
cells were cryopreserved by putting
them into standard3.6-ml cryotubes
(Nalge Nunc) at 2 X 106 cells per
ml in 2 ml with final
concentrations of 10% dimethyl
sulfoxide and 10% autologous plasma
while being maintained at 4C.
These tubes were then placed into
beakers with methanol, which were
placed into -70C freezers. After
24 h, the frozen samples were
placed into liquid nitrogen. where
they remained until being removed

for assay, After thawing, cells were

either used without further separation
or were further subdivided by
fluorescence-activated cell sorting
(FACS; Becton Dickinson) into CD34+,
CD34+CD38-, and CD34+CD38+ populations
(each 2:98% CD34+) or by magnetic beads
into CD34+ cells (2:95% CD34+) as
described (31-33).
In Vitro Colony Assays for HPC.
Prefreeze and defrosted cells were
plated at two to three different
concentrations each to yield high
enough colony numbers to get accurate
colony counts with a
Abbreviations: BFU-E, burst-forming
unit-erythroid; CFU-GM, colonyforming
unitgranulocyte, -macrophage; CFU-GEMM,
colony-forming unitgranulocyte,
-erythrocyte, -monocyte, and
-megakaryocyte; C8, cord blood; HSC,
hematopoietic stem cell; HPC,
hematopoietic progenitor cell; NOD,
nonobese diabetic; SOD, severe combined
immunodeficient; rhu, recombinant
human; GM-CSF, granulocyte-macrophage
colony-stimulating factor; SLF, steel
factor; Epo, erythropoietin; FL, Flt3ligand.
'To whom correspondence should be
addressed at: Walther Oncology Center,
Indiana University School of Medicine,
1044 West Walnut Street, R4-302,
Indianapolis, IN 46202.
Email: hbroxmey@iupui.edu.
PNAS I January 21, 2003 I vol. 100 I
no. 2 I 645-650

minimal of colony overlap. Cells

were plated in 1 % methylcellulose
culture medium with 30% FBS
(HyClone) and with the following
mixture of growth factors [1
unit/ml recombinant human (rhu)
erythropoietin (Epo; Amgen
Biologicals), 10 ng/ml rhu IL-3
(Immunex), 10 ng/ml rhu
granulocyte-macrophage colonystimulating factor (GM-CSF,
Immunex), and 50 ng/ml rhu steel
factor (SLF, also called stem cell
factor; R & D Systems), or 1
unit/ml rhu Epo plus 10 ng/ml rhu
IL-3] for analysis of granulocytemacrophage [colony-forming unitgranulocyte, -macrophage (CFU-GM)],
erythroid [burst-forming uniterythroid(BFU-E)], and CFU-GEMM
progenitors, or in 0.3% agar
culture medium with 10% FBS, 10
ng/ml rhu GM-CSF alone, or with 50
ng/ml rhu SLF with or without 100
ng/ml rhu FIn-ligand (FL, Immunex)
for analysis of CFU-GM. Colonies
were scored after 14 days
incubation in a humidified chamber
at 5% CO2 and lowered (5%) O2.
Details regarding the assays, what
they measure, and the exact
ingredients used have been
published (34).
Secondary Replating of CFU-GEMM.
Replating of CFU-GEMMcolonies from
a 1 to a 2 dish are considered a
means to estimate the "selfrenewal" capacity of CFU-GEMM and
were performed as described (24)
but without the use of CB plasma,
which is known to enhance detection
of replatable CFU-GEMM (25).
Individual CFU-GEMM colonies that
grew in methyl cellulose plates
after 14 days incubation with Epo,
IL-3, GMCSF, and SLF were removed,
each colony made into a single cell
suspension and plated in a single
2 plate in methylcellulose culture
mixture containing the same
ingredients and growth factors as
in the primary culture. Assayable
2 colonies were scored as CFU-GM,
BFU-E, or CFU-GEMM 14 days later.

Ex Vivo Expansion. Frozen CB samples

were thawed and washed with complete
medium (CM, consisting of Iscove's
modified Dulbecco's medium supplemented
with 10% FCS, 1 % L-glutamine and
penicillin and streptomycin). Cells
were stained with CD34 FITC and CD38
phycoerythrin [PE; Becton Dickinson
Immunocytometry Systems (BDIS)], and
total CD34+ cells or subfractions of
CD34+ cells were isolated by flow
cytometric cell sorting by using a
FACSYantage SE (BDIS). Sorted cells
were cultured ex vivo in individual
wells of 24-well plates of CM
supplemented with FL and megakaryocyte
growth and differentiation factor
(MGDF, also called thrombopoietin) at
50 ng/ml each; SLF, IL-3, and IL-6 at
100 ng/ml each; and GM-CSF at 20 ng/ml.
Cytokines used for ex vivo expansion
were a kind gift from Amgen
Biologicals. Cytokines were
supplemented at the initiation of
culture and every 48 h thereafter.
Cultures were demidepopulated every 710 days, and care was taken not to
allow cell numbers to exceed 106 cells
per ml. Cells recovered from culture at
every time point were counted and
plated in clonogenic assays as
described above. Data are reported as
cumulative cell or progenitor cell
production in culture after correcting
for the number of cells removed after
each demidepopulation as described
(35). Results were compared with the ex
vivo expansion capability of nonfrozen
freshly isolated CB collected from
different donors than those used for
the frozen samples, because prefreeze
data on the frozen cells was not
available.
f)lOD/SCID Mouse Assay. CD34 + cells
were isolated by magnetic beads from
defrosts of lS-yr frozen CB samples and
assayed as detailed (31). Between 2 X
104 and 3 X 104 CD34 + cells were
isolated from each donor sample
separately, and the purified cells
(2:95% CD34 +) from each donor sample
were injected into two sublethally
irradiated NOD/SCID mice whereby each
recipient received between 1 X 104 and
1.5 X 104 CD34+ cells.

These NOD/SCID mice were given 300

rad from a 137CS source, and cells
were injected into mice 2 h later.
Controls were aged-matched NOD
/SCID mice that received no
irradiation or cells. NOD/SCID mice
were purchased from The Jackson
Laboratory.
646 I
www.pnas.orgjcgi/doi/l0.1073/pnas.0
237086100
'~
~a Table 1. Percent recovery of
cryopreserved CB cells after 15 and
I il 10 yr J Parameter, yr Mean 1
SD Range No. ~1 f Nucleated cells
15 83 12 64-100 9 10 88 20 47100 10 CFU-GM 15 95 15 56-100 9
10 92 11 81-100 10 BFU-E 15 84
25 29-100 9 10 91 17 55-100 8
CFU-GEMM 15 85 25 29-100 9 10 74
25 40-100 8
Data from the 10-yr defrost were
reported (23) and are shown here
for comparison.
Results
Recovery of Viable Nucleated Cells
and HPC. We had previously
demonstrated high-efficiency
recovery of immature and more
mature subsets of HPC from human CB
that had been cryopreserved for up
to 10 yr (23). In that study,
recovery of nucleated cells
averaged 88%, whereas 74-92%
recovery of HPC was found; some
samples in each category were
recovered at 100% efficiency. This
was a direct comparison of the same
samples assayed prefreeze and
postthaw after 10 yr by using the
same culture conditions and growth
factors. In the present stud)!, we
also compared recovery of nucleated
cells and HPC after thawing of the
exact same frozen donor samples
evaluated after 10 yr by using the
same prefreeze cytokines and growth
conditions (Table 1). There was no
statistical difference in the
recovery after 10 and 15 yr
compared with prefreeze values of

nucleated ceBs, or of CFU-GM, BFU-E, or

CFU-GHvlM that were detected by colony
formation stimulated by the combination
of Epo and IL-3 in methylcellulose
cultures or of CFU-GM that were
detected by colony formation stimulated
by Glvl-CSF in agar cultures. As with
the 10-yr defrost, we found up to 100%
recovery of the prefreeze nucleated
cell and colony numbers.
When the original cells were frozen,
potent costimulating factors such as
SLF (36) and FL (37) had not yet been
identified.
The addition of SLF or FL with a CSF
allowed detection of more immature
subsets of HPC with a higher
proliferative capacity than was noted
with cells stimulated with CSFs in the
absence of SLF or FL (16, 36, 37). By
the time we analyzed the 5- and lO-yr
defrosts, we were also able to assess
the numbers and size of colonies formed
from defrosted cells cultured with
growth factors in the presence of SLF;
the recovered HPC manifested a very
high proliferative capacity after
stimulation with SLF plus a CSF such as
Epo or GM-CSF (16, 23). A direct
comparison of 10- vs. 15-yr defrosts is
shown in Table 2 for CFU-GM responsive
to stimulation by either GM-CSF, GM-CSF
plus SLF (in agar), or Epo,IL-3 plus
SLF; for BFU-E responsive to
stimulation by Epo plus IL-3; and CFUGEMM responsive to stimulation by Epo,
IL-3 and SLF (in methylcellulose). As
noted previously (16), CFU-GEMM, but
not BFU-E, colonies are seen when SLF
is added with Epo plus IL-3 (clata not
shown). There were no significant
differences in the retrieval of HPC
(responsive to stimulation with a CSF
or CSFs plus SLF) after 10 and 15 yr.
Additionally, we were able to
quantitate the retrieval of CFU-GM
stimulated with FL in combination with
GM-CSF, in the absence and presence of
SLF, although we could not directly
compare this to prefreeze or 5- or 10yr defrosts of these same samples, as
we had not used FL in the previous
studies.
Defrosted CB HPC stored frozen for IS
yr had extensive
Broxmeyer et al.

Table 2. Recovery of immature HPC

from cryopreserved CB after 15 and
10 Yr, expressed per 106 cells
frozen
Parameter, yr
Mean:!: SD.
Range
No.
CFU-GM colonies stimulated with GMCSF 1 5 318 :!: 217 140-840 10
101 :!: 83 16-234 CFU-GM colonies
stimulated with GM-CSF + SLF 15
1621 :!: 833 380-3441 10 1501 :!:
746 545-2565 CFU-GM colonies
stimulated with GM-CSF + FL 15
1513:': 733 380-3159 10 ND CFU-GM
colonies stimulated with GM-CSF +
FL + SLF 15 2214 925 860-4400 10
ND CFU-GM colonies stimulated with
Epo + IL-3 + SLF 15 1329 479 4402160 10 1710 902 669-3116 BFU-E
colonies stimulated with Epo + IL-3
15 1235655 160-2119 10 627351
212-1178 CFU-GEMM colonies
stimulated with Epo + IL-3 + SLF 15
2018:!: 1025 280-3480 10 1390 :':
1004 378-3254
ND, not done.
proliferative capacity as assessed
by the number of cells contained in
individual colonies. The results
given are the average results of 40
BFU-E, 40 CFU-GEMM, and 34 CFU-GM
colonies each assessed separately
from a total of defrosts from seven
different donors. BFU-E stimulated
with Epo and IL-3 gave rise to
39,000 29,000 ( SD; range
10,000-182,500) erythroid cells per
colony.
CFU-GEMM stimulated with Epo, IL-3
and SLF gave rise to colonies
containing 198,100 52,900 (range
70,000-292,500) erythrocytes,
granulocytes, macrophages, and
megakaryocytes, although not all
colonies contained megakaryocytes.
CFU-GM stimulated with the

combination of Epo, IL-3 and SLF gave

rise to colonies containing 12,100
5,400 (range 2,500-22,500) granulocytes
and macrophages. However, the
proliferative capacity of these CFU-GM
is likely a large underestimate of the
true proliferative capability of these
cells; CFU-GM colonies in methyl
cellulose were not nearly as large as
those growing in agar culture
stimulated with GM-CSF plus SLF, with
or without FL. We did not count the
number of cells contained in individual
colonies formed in agar as this is more
technically difficult to do accurately.
However, actual sizes of the largest
colonies, as visualized in the
semisolid medium plates, can be seen in
Fig. 1 (CFU-GM colonies stimulated with
GM-CSF, GM-CSF plus SLF, GM-CSF plus
FL, or GM-CSF plus SLF and FL). As
previously reported for
noncryopreserved cells (36,37), CFU-GM
colonies stimulated with GM-CSF plus
either SLF or FL are larger than those
stimulated with only GM-CSF, and the
addition of SLF plus FL to cultures
with GM-CSF yields the largest colonies
(37). The ratios of CFU-GM colonies
that formed from the 15-yr defrosts (n
= 9) in the presence of GM-CSF plus
SLF, GM-CSF plus FL, or GM-CSF, SLF and
FL compared with those stimulated only
by GM-CSF were, respectively, 6.1 3.3
(mean 1 SD; range 2.6-12.4), 5.8
3.0 (2.6-11.9), and 8.5 3.6 (4.715.0). Thus, CFU-GM subsets from
defrosted CB, similar to that of fresh
noncryopreserved CB, that had greater
proliferative capacity were in greater
quantity than those with lesser
proliferative capacity. Visualization
of the actual largest representative
BFU-E colonies formed in
methylcellulose with Epo and IL-3, and
representative CFU-GEMM colonies in
methylcellulose with Epo, IL-3 and SLF
are shown in Fig. 1. The sizes of these
colonies are similar to those shown for
the 10-yr defrosts stimulated with the
same
Broxmeyer et al.
CFU-GM Colonies
9 8

9 8
9
9
GM-CSF
9 8
BFU-E Colonies
~'t
'4
"
, ."
CFU-GEMM Colonies
~
9 8
.. ~"
.V
.~ <11> ~
~ *'
.. rg q
. ~
'II;
."
~ ~ ~
. ..
.' .
.
9 8
Epo+IL-3
Epo+IL-3+SLF
Fig. 1. Colonies formed from CB
CFU-GM, BFU-E, and CFU-GEMM stored
frozen for 15 yr, defrosted, and
plated in semisolid agar culture
medium for 14 days in the presence
of GM-CSF alone or in combination
with SLF, FL, or SLF plus FL for
CFU-GM, and in semisolid methyl
cellulose culture medium for 14
days, respectively, in the presence
of Epo plus IL-3 and of Epo and IL3 plus SLF for BFU-E and CFU-GEMM.
These are representatives of the
largest colonies formed. Numbers of

these colonies per 106 defrosted cells

can be found in Table 2.
cytokine combinations (23) and to those
of freshly obtained CB.
The above results demonstrate highefficiency recovery of HPC with
extensive proliferative capacity after
15-yr storage in a frozen state.
Recovery of CFU-GEMM with Replating
Capability. In previous studies, we had
shown that CFU-GEMM colonies from
nonfrozen CB could be replated as
single colonies per plate with the
formation of 2 CFU-GEMM, BFU-E, and
CFU-GM colonies (24, 25). Many of the
2 CFU-GEMM colonies were as large as
the 1 CFU-GEMM colonies from which
they were derived, suggesting a degree
of self-renewal capacity for CFU-GEMM.
In the present study, for the first
time, we evaluated the capacity of
cryopreserved CFU-GEMM to generate 2
colonies of CFUGEMM, BFU-E, and CFUGEMM (Table 3). The CFUGEMM present in
defrosts of 15-yr frozen CB had
extensive replating characteristics. In
fact, 96.6% of the 2 plates contained
at least one colony that was either a
CFU-GEMM, BFU-E, or CFU-GM. More
impressively, 37.9% of the 2 plates
contained at least one CFU-GEMM, and
every plate that contained a 2 CFUGEMM colony also contained BFU-E and
CFU-GM colonies. On average, every 1
CFU-GEMM colony gave rise,
respectively, to 37.3 (range 0-98),
14.1 (range 0-166), and 2.0 (range 018) CFU-GM, BFU-E, and CFU-GEMM
colonies per 2 plate. Because we did
not add CB plasma to either the 1 or
2 plates, and CB plasma enhances the
replating efficiency of 1 CFU-GEMM
(25), it is likely that we have
underestimated the
PNAS I January 21, 2003 I vol. 100 I
no. 2 I 647

c
Table 3. Percent secondary
replating capability of single CFUGEMM retrieved from 15-yr frozen CB
Any progenitor CFU-GM, BFU-E, and
CFU-GEMM CFU-GM and BFU-E CFU-GM
and CFU-GEMM BFU-E and CFU-GEMM
CFU-GM BFU-E CFU-GEMM
Results are based on 29 replated
colonies from a total of six
different donors. Both l' and 2'
colonies were plated in
methylcellulose culture medium with
Epo, IL-3, and SLF. At least one
secondary colony was detected in 28
of the 29 primary colonies plated.
replating (self-renewal) capacity
of individual CFU-GEMM from frozen
CB. Although we did not do
replating evaluations on the
original CB samples used in the
present study, the replating
capacity seen is similar to that of
fresh CB (24). Thus, these results
demonstrate the retention of selfrenewal capacity of CFU-GEMM after
15-yr storage in a frozen state.
Recovery of HPC/HSC That Can Be
Expanded ex Vivo. Numerous
investigators have shown the
extensive capacity of unfrozen CB
HPC/HSC for ex vivo expansion
(reviewed in refs. n, 16, and 3539). This extensive expansion
capability has also been seen with
CB stored frozen for short periods
of time (26-28). To assess the
effects of longer storage of frozen
CB, we evaluated the capacity of CB
frozen for 15 yr to give rise, in
suspension cultures supplemented
with growth factors, to nucleated
cells ane! !-IrC in longterm
cultures (Fig. 2). Over the course
of35 days in cullure, freshly
isolated (control) and
eryopreserved CB CD34" cells
supported the
WOiIksI., CultufD
3 4 WGollaIn Culturo

o
140000
350
ea2 (C034+)
...
C63{CDJ.4+)
"'"
CB2tJ.4"~1
,..,
C83(34":-)
~
C22(eO)-h)
...,
eBl (COJh)
""
c".a~~"'Ja.1 CB3{:w.~3-J .<J.
'a~ 120000 .2 ~ 1000~O ~ ~ 80000
! i 600.' o z 40000 u., ~ 20000
o Q 300 ~ ~ 250 in:;
-= u 200 ~!150 ~ ~ 100 50
, 3 4
Weeks In Cult.UfO
, 3 4
Weeks I" Culture
Fig. 2. Fold expansion in total cell
number and donogenic progenitors in
long-term cultures of CD34+ or
CD34+CD38- cells from fresh (control)
or cryopreserved CB samples. CD34+
cells from fresh CB samples or CD34+
and CD34+CD38- cells from cryopreserved
CB samples were expanded in longterm
cultures as described in Materials and
Methods and were demidepopulated every
7 to 10 days. The total cell number
contained in each culture at
differenttime points was calculated by
using the formula X = (number of cells
per culture) x (1/2)" where X is the
number of total cells in culture and n
is equal to the number of previous
demidepopulations. The total number of
cells at different time points was
divided by the number of cells used to

initiate each culture to calculate

fold expansion of cells (A and C).
The same formula was used to
calculate the total number of
clonogenic cells produced in
culture, and, similarly, fold
expansion of total progenitors was
determined (8 and 0).
CD34 + cells from a total of four
cryopreserved CB samples and two
fresh samples were used in these
studies, and long-term cultures
were initiated with CD34+CD38cells from two of the four
cryopreserved samples.
648 I
www.pnas.org/cgi/doi/10.1073/pnas.0
237086100
--- ---96.6 37.9 79.3 37.9 37.9 93.1 87.8
37.9
Table 4. In vivo repopulation of
mouse NOD/SCID bone marrow with
CD34 + cells isolated from CB cells
stored frozen for 15 yr
Sample % CD45+ % CD45+ ICD19+ %
CD45+ ICDll b+
Untransplanted 0.04 0.1 0.01
control 0.02 0.05 0.02 0.01 0.12
0.01 1 0.3 1.2 7.4 la 0.03 0.0 0.0
2 2.4 68.1 19.3 2a 0.5 49.7 10.2 3
1.5 5.7 4.1 3a 0.8 20.6 5.6 4 1.44
42.3 16.3 4a 0.5 58.0 25.2
Percentages shown are for human
cells in the windows shown in Fig.
3 set on the CD19 or CD 11 b
isotype at a background level of 1
%. Sample numbers reflect two
recipients (e.g., 1, la or 2, 2a,
etc.) of each CB donor. Mice 1, 2,
3, and 4 were evaluated 11 wk after
injection of cells into NOD/SCID
mice. Mice 1 a and 2a were
evaluated 13 wk after injection,
and m;c" 'la and 3b were evaluated
12 wk after injection.
production of> 1,000- to J 3,000foJd higher numbers of cel!s than
used to initiate the long-term

cultures (Fig. 2A). Kinetics of

expansion of assayable progenitors in
cultures initiated with CD34 + cells
from cryopreserved samples were also
similar to those observed in cultures
initiated with control cells, whereby,
at peak production in all cultures, in
excess of 10- to 2S-fold more
progenitor cells were detected relative
to the initial number of clonogenic
cells contained in each culture (Fig.
2B). .
CD34+ cells can be subphenotyped with
the CD38 marker into populations of
more immature (CD34+CD38-) and more
mature (CD34+CD3R+) HPe, with HSC found
in the CD34+CD3Spopulation. CD34'CD38and CD34+CD38+ cells were isolated from
cryopreservee! CB samples, and longterm cultures were initiated with both
phenotypes of cells. Both total cell
production and expansion of progenitor
cells were considerably higher among
CD34+CD38- cells than their CD38 jcounterparts (data not shown). Fold
expansion of both total cells (Fig. 2C)
and clonogenic cells (Fig. 2D) were
substantially higher in cultures
initiated with CD34 +CD3S- cells
compared with similar cultures
originated with CD34 + cells from the
same cryopreserved CB samples,
confirming the primitive nature and
extensive proliferative potential of
CD34 +CD3S- cells. Fold increase in the
number ofHPC in cultures initiated with
CD34+CD3S- CB cells exceeded ISO in
both long term cultures.
Recovery of NOD/SCID Repopulating Human
HSC. To assess the cryopreservation of
CB HSC more directly then what is
described above, which mainly examines
the activity of HPCs, we took advantage
of the NOD/SelD mouse as an assay for
engrafting and repopulating human stem
cells (29-32, 39).
Magnetic bead separated CD34 + cells
isolated from defrosted cells of four
donors were each injected into two
NOD/SelD mice. Mice were evaluated for
human cell engraftment 11-13 wk after
injection of CB cells. As shown in
Table 4 and Fig. 3, human NOD/SCID
repopulating cells were present in the
four samples, and were noted in both
recipients of each donor cell inoculum

in three of the four samples tested

as determined by the percentage of
CD45+, CD45+ /CD19+, and CD4S/CDllb+ human cells in mouse bone
marrow. Engraftment ofNOD/SClD mice
11-13 wk after injection of human
cells is considered a measure of
relatively long-term engrafting
human stem cells (32). The low
percentage of human cells
engrafting these mice is due to the
limiting numbers of CD34 + cells
available for transplantation in
the NOD/SCID mice, because we
wished to assess the repopulating
capacity of single collections of
CB.
Broxmeyer et al.

~ Mous.'
L,~~~J. Mous.4.
Mouse 2
10 101 10 10 101
;.[;j Untransplanted . .. Control
#3
,
100 10' 102 10 104
CD19 APC
CD11b PE
Fig. 3. Flow cytometric analysis of
human CD45' cells with CD19 and
CD11 b surface phenotypes found in
the bone marrows of NOD/SCID mice 4
mo after infusion of bead-separated
CB obtained from defrosts of cells
cryopreserved and stored frozen for
15 yr. The hi,tograms are of cells
that express human CD45 above
background levels (set at 1 % of
isotype). The actual percentages of
human cells that are CD45+,
CD45+CD19+, and CD45+CDll b+ are
reported in Table 4.
However, the level of human
chimerism in these mice is
consistent with that seen
previously (31) by using similar
low numbers of transplanted fresh
CD34 + cells. These results
demonstrate that CD34+ cells
isolated from CB stored frozen for
IS yr have the capacity to engraft
the marrow of NOD/SCID mice at a
frequency equivalent to that
previously shown for freshly
isolated CB CD34 + cells.
Discussion
CB transplantation is a relatively
recent clinical procedure, with the
first CB transplant performed in
October 1988 (1, 11, 19).
Since then, hundreds of thousands
of CB collections have been stored
frozen throughout the world, in

anticipation of their potential use to

treat a multitude of malignant and
nonmalignant disorders in children and
adults. However, the longest that a CB
collection has been in frozen storage
before actual clinical use is likely
between 3 and 5 yr. Our own studies
have previously demonstrated highefficiency recovery of immature and
mature subsets of CFU-GM, BFU-E, and
CFU-GEMM after 5 (16) and 10 (23) yr of
frozen storage. These recovery
calculations were possible because of
direct comparisons of post- and pre
freeze HPC numbers obtained from the
same CB samples using the exact same
culture conditions. Since our last
report in 1997 (23), papers published
in 1998 and 1999, respectively,
described results of analysis of 15and 12-yr defrosts of CB. The 15-y r
study (40) reported on recovery of
relatively mature subsets of CFU-GM (as
detected by colony formation after
stimulation with 5637 conditioned
medium), but, because no prefreeze data
were available for these same samples,
the efficiency of recovery of the CFUGM could not be calculated. The 12-yr
study (41) reported recovery of CFU-GM,
BFU-E, and CFU-GEMM based on comparing
numbers of cells to 1-mo defrost values
from the same samples. These authors
noted >90% recovery of HPC after 10 yr.
Because the recovery calculations were
not based on prefreeze control numbers,
the actual efficiency of recovery could
not be absolutely calculated. Although
it is clear from the above studies that
one could retrieve viable HPC, there
was the reasonable and more important
concern of whether or not the more
immature HSC population survived the
freezing procedure and long-term
storage.
Broxmeyer et al.
The practical aims of the present study
were to evaluate recovery of HPC after
15-yr storage in a frozen state, and
most importantly to determine not only
the in vitro proliferative, selfrenewal and expansive properties of
recovered HPC, but also to evaluate the
capacity of recovered cells to engraft
and repopulate the hematopoietic system

of sublethally irradiated NOD/SCID

mice. Our results clearly
demonstrate that HPC with extensive
proliferative, self-renewal and ex
vivo expansion capabilities, and
HSC with NOD/SCID repopulating
ability can be effectively
recovered after 15-yr storage in a
frozen state. As part of om initial
preclinical studies that suggested
the feasibility of CB
transplantation (14), we have had
CB samples stored frozen since the
mid] 980s. HPCs responsive to the
stimulatory effects of combinations
of cytokines, including CSFs (Epo,
GM-CSF, and IL-3) and the potent
costimulating cytokines (SLF and
FL), are considered more immature
than HPC responsive to single
cytokines (e.g., GM-CSF, IL-3, or
Epo), conclusions supported by the
more extensive proliferative
capacity of HPC responsive to
stimulation by multiple vs. single
cytokines. In fact, some of the HPC
responsive to stimulation by
multiple cytokines including SLF
and/or FL may actually reside
within the HSC compartment (16, 24,
25). That single colonies from CB
CFUGEMM in such culture conditions
can be replated in 2 dishes' with
generation of CFU-GEMM, BFU-E, and
CFU-GM colonies is highly
suggestive of the self-renewal
capacity of these CB CFU-GEMM.
Self-renewal capacity is a hallmark
of stem cells.
Whereas assessment of CFU-GEMM
activity in vitro may not represent
long-term repopulating stem cell
function, the data from these
studies are consistent with these
cells being a subset, although a
more mature subset, of HSC. Because
the replating capacity of IS-yr
defrosts of CB CFU-GEMM is as
extensive as that of freshly
isolated CB CFU-GEMM, and the
recovered CD34 +CD38- p(Jpulation
had the capacity, at least as
potent as such fresh cells, to be
ex I'ivo expanded, it was clear
that very early cells within the
HSC/HPC compartments had withstood
the cryopreservation and storage

conditions used. However, ultimately,

whether or not any stem ceil
collection, whether from a freshly
obtained or frozen sample, will be
useful for clinical transplantation
depends on the ability of the cells to
appropriately home to a
microenvironment niche conducive for
their self-renewal, growth, and
differentiation. Whether this homing
has occurred is usually measured by the
long-term engrafting capability of the
infused cells. Whereas assays for HSC
homing/ engraftment have been well
established for decades with rodent
cells, it is only recently that assays
that apparently detect such human HSC
have become available. The most common
and widely used assay for human HSC
engrafting capabilities is the human
mouse SCID repopulating cell assay (2932). In fact, this in vivo assay has
shown that human CB HSC are more
effective than their bone marrow
counterparts in engrafting SCID mice,
results consistent with the lower
number of CB, compared with adult bone
marrow cells, needed for clinical
engraftment (1-13, 17-18,20,22). Our
data herein show that 15-yr defrosts of
frozen CB engraft and repopulate the
hematopoietic system of NOD/ SCID mice
in a quantitative and qualitative
manner consistent with that of freshly
isolated CB cells.
Whereas final proof for the long-term
engrafting capability of CB
cryropreserved and stored frozen [or
long periods of time requires clinical
results demonstrating long-term
engraftment in humans, our inclusive
results are highly suggestive that CB
can be stored frozen for at least IS yr
with highly efficient recovery of
viable and highly functional HSC and
HPC needed for successful CB
transplantation.
These studies were supported by U.S.
Public Health Service National
Institutes of Health ROI Grants RUt
HL56416, ROI HL67384, and ROI DK53674
from the National Institutes of Health
(to I-LE.B.) and ROl I-IL55716 from the
National Heart, Lung, and Blood
Institute (to E.F.S.).

PNAS I January 21, 2003 I vol. 100

I no.2 I 649

c
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