Early Human Development (2007) 83, 733–741

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / e a r l h u m d e v

Cord blood revelations—The importance of being a

first born girl, big, on time and to a young mother!
C.P. McGuckin ⁎, C. Basford, K. Hanger, S. Habibollah, N. Forraz

Newcastle Centre for Cord Blood, Institute of Human Genetics, Newcastle University, NE1 3BZ Newcastle upon Tyne,
United Kingdom

KEYWORDS Abstract
Cord blood;
Stem cells; Umbilical cord blood (UCB) has become an alternative source for providing haematopoietic stem/
Obstetric history; progenitor cells as well as non-haematopoietic stem cells, compared to the conventional sources
Birth weight; of bone marrow (BM) and peripheral blood (PB). Although UCB has many advantages over BM and
T-cells; PB there are still limitations to its use in the clinical setting, principally cell numbers. Thus, this
B-cells; study aimed to characterise components that comprise UCB samples and the physiological factors
Dendritic cells that affect them: (i) gender, (ii) obstetric history, (iii) infant birth weight, (iv) gestation stage and
(v) mother’s age.
Our results show that UCB total nucleated cell (TNC) and haematopoietic stem cell (CD45+/CD34+)
content is significantly affected by the baby’s birth weight, mother’s age at delivery, mother’s
obstetric history, and gestational stage at due date, all with p valuesb 0.0001. The only parameter not
found to be significant was gender, although results did suggest that female infants provide greater
stem cell numbers than their male counterparts. Other UCB cellular sub-types affected were T-cells,
dendritic cells and B-cells.
In conclusion, this study demonstrates that many different obstetric factors must be taken
into account when processing and cryo-banking UCB units for transplantation.
© 2007 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
In recent years umbilical cord blood (UCB) has widely become
an additional source of stem cells for transplantation for a
variety of haematological and non-haematological malignan-
cies and disorders. Conditions such as leukaemia e.g. acute
myeloid leukaemia (AML) [1], metabolic disorders such as
Krabbe’s Disease [2] and immune deficiencies such as Wiskott
Aldrich Syndrome (WAS) [3] have all been successfully treated
⁎ Corresponding author. Tel.: +44 191 241 8824.
E-mail address: c.mcguckin@ncl.ac.uk (C.P. McGuckin).
with UCB transplantation. Since the first UCB HSC transplant
in 1972 [4] over 6000 similar UCB transplants have taken place
worldwide [5]. An easily available, viable alternative to bone
marrow (BM) and peripheral blood (PB) which provides stor-
age of units from ethnic minorities not normally possible
within BM donor registries [6] and allows for an increase in the
rate of matched unrelated donor allogeneic transplants. [7].
A lower risk of the graft versus host disease (GvHD) after
transplantation of UCB when compared to BM [8] has also
been documented. This could be due to the fact that cells
transplanted from UCB are more naïve and have lower human
leukocyte antigen (HLA) protein expression [7]. It was also
demonstrated that UCB lymphoid progenitors yielded a more

0378-3782/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.earlhumdev.2007.09.001
734
efficient thymic regeneration pathway despite the low num-
ber of cells infused compared to BM, arguing for a complete
clinical immune recovery after UCB transplantation [8,9].
Other studies have also shown that as well as being a source
of haematopoietic progenitor cells, UCB also contains non-
haematopoietic stem or progenitor cells including mesench-
ymal and endothelial precursors [10].
Although a valuable source of HSCs, in order to effectively
bank UCB units suitable for transplantation, samples need to
be characterised and obstetric factors which impact upon
UCB quality should be further examined. In this study five
different physiological parameters that pertain to either the
baby or the mother were compared with levels of various
UCB cellular sub-types such as HSC, dendritic cells (DC) and
T-cells and TNC. The UCB TNC was calculated as the pre-TNC
count divided by the UCB’s pre-start volume. The five
different parameters were baby’s gender, baby’s birth
weight, mother’s age at delivery, obstetric history and
gestational stage at parturition (a standard nine month
pregnancy equals 40 weeks).
Previous work shows that some patterns have already
emerged. Birth weight of the infant positively correlates
with TNC [11–13]. When looking at maternal age, a previous
study demonstrated that term and pre-term UCB contain a
significantly higher number of early and committed pro-
Figure 1 A list of human CD antigens, which cells they are expressed on, and a brief description of their functions.
C.P. McGuckin et al.
genitor cells and that they are better able to form colony-
forming unit-granulocyte-macrophage (CFU-GM) when com-
pared to adult PB [14]. Infant gender has previously been
found to have an impact on TNC of UCB samples and
newborn boys appear to have fewer stem cells than girls
[12,15].
UCB is also a useful source of stem cells for further
scientific research. Our group has shown that a naïve sub-
population of these cells, known as cord blood embryonic-
like stem cells (CBEs), have many of the same properties
as embryonic stem cells [16] but without the controversy
attached. Studies from our research group and others
showed that it is possible to manipulate UCB stem cells in
the laboratory and direct them to differentiate into a num-
ber of tissues including neural, hepatic or pancreatic-like
cells [17]. UCB stem cells can for instance be combined to
biomaterials and growth factors to engineer 3-dimensional
human tissue constructs for pharmaceutical testing and to
further develop clinical protocols for regenerative medi-
cine applications.
The major challenge associated with UCB is cell content. It
has been suggested that an average TNC for an UCB transplant
should be a minimum of 2 × 107 cells per kg of body weight,
quite a challenge for an adult patient [5]. Yet, although the
TNC of UCB can be limited, it has been shown to contain a
Cord blood revelations—The importance of being a first born girl, big, on time and to a young mother! 735

Figure 1 (continued ).

higher frequency of early progenitor cells than PB or BM [18].
The principle aim of this study was to optimise UCB separation
and cryo-preservation by the characterisation of these cel-
lular groups. Several physiological factors were examined
in order to determine the most suitable method. However,
some of these findings appeared to be of particular interest
themselves.
2. Materials and methods
2.1. Inclusion criteria
Samples were collected, only after written and informed
consent by the parents, from the Maternity Unit at the Royal
Victoria Infirmary, Newcastle upon Tyne. A negative viral
736 C.P. McGuckin et al.

sample collection is complete, the unit is transported back to

the laboratory and stored at 4 °C until processing is initiated.

2.3. Laboratory processing

Before the processing of each unit of UCB, using methods

Figure 2 HSC sub-types and their surface markers. Differen-
tiation of HSC occurs in a specific order from early stage to mid-
stage and finally late stage. These stages are defined by their
surface antigen expression. CD45+dim/CD34−/CD133+ → CD45+/
CD34+/CD133+ → CD45+/CD34+/CD133−.
profile was also required. UCB units were processed only if
the time between collection and cryo-preservation was
b24 h and the sample volume was ≥ 50 ml.
including red cell depletion and density gradient separation,
a sample was removed for flow cytometric analysis and to
ascertain the TNC. Differential counts were performed on
the CellDyn4000 haematology analyser. FACS analysis was
performed on the BD FACS Caliber and analysed using Becton
Dickinson (BD) FACS software Cell Quest Pro. Red blood cells
(RBC) were lysed using PharmLyse solution (BD, Cat 555899,
Lot. No. 56213 Oxford, UK) and washed on the BD lyse/wash
machine. Surface markers quantified are detailed in the
table below (Fig. 1).
2.4. Statistical analysis

2.2. Collection
The samples were collected from both vaginal and caesarean
section births. The samples were collected post partum,
once the placenta had left the mother. The umbilical cord
was clamped in two places; close to the placenta and close to
the baby. The placenta was then hung in a cone shaped
collection vessel with the cord hanging down through the
bottom. Collection bags used contain citrate phosphate
dextrose adenine (CPD-A) anticoagulant and have a needle
attached (Baxter Healthcare PL146-CPDA-1-35ml, Newbury,
UK). This is spiked into the cord at the bottom allowing the
blood to drain into the collection bag. Only blood from the
umbilical cord is collected, not from the placenta. Once the
Statistical tests were performed with the software program,
Prism (GraphPad, Version 3, San Diego, USA). Data were
analysed using non-parametric, two-tailed Mann–Whitney
U-testing. A p b 0.05 was considered to be statistically
significant.
3. Results
In order to fully define the UCB cellular sub-populations, HSC
were divided into three distinct groups according to a model
previously described [19] from primitive to mature stem cells
(Fig. 2).
Results are presented according to five different obstetric
factors: (i) baby’s gender, (ii) baby’s birth weight, (iii)

Figure 3 Effects of gender on different UCB cell populations. A. UCB T-cell (CD34+/CD3+) populations are significantly higher in
female infants compared to male (p b 0.001). B. UCB TNC in relation to gender. The average TNC for the 20 males was 10.5 (± SD 2.18)
whereas the female (N = 23) average TNC was higher at 11.5 (± SD 0.77). However, upon statistical analysis the difference was not found
to be significant (p = 0.75). C. Graph C illustrates that females have a slightly higher primitive HSC (CD45dim/CD34−/CD133+)
concentration than males although the correlation is not significant with a p value = 0.89. D. Graph D shows that females have a higher
mature HSC (CD45+/CD34+/CD133−) concentration than males and are again not significant with a p value = 0.95. n = 43.
Cord blood revelations—The importance of being a first born girl, big, on time and to a young mother! 737

mother’s age at delivery, (iv) obstetric history and (v)
gestational stage at parturition.
3.1. Infant gender
The cohort for this study was a total of 43 samples with a
distribution between the sexes of 20 males (47%) and 23
females (53%). When observing T-cell (CD34+/CD3+) popula-
tions (Fig. 3A) it was found that females have a greater
concentration of this cell type than male infants. The
correlation was shown to be significant with a p value of
b 0.001. Upon comparing average UCB TNC to the gender of
the baby it was observed that females tend to have a slightly
higher UCB TNC than males (Fig. 3C), although the difference
was not statistically significant (p 0.75). When looking at
HSC, findings indicate that females have higher concentra-
tions of early (CD45+dim/CD34−/CD133+), and late stage
(CD45+/CD34+/CD133−) sub-types.
3.2. Obstetric history
Data were collected on obstetric history of the mother i.e. the
total number of pregnancies a mother had had (even if no live
birth resulted). In this study the number of pregnancies ranged
from 1 to 7. This parameter impacts upon UCB TNC (Fig. 4B),
with each additional pregnancy the UCB TNC decreases
significantly (p b 0.0001). When looking at early stage HSC
populations (Fig. 4B), dendritic cells which express MHC class II
surface antigens (Lin1−/CD11c+/HLA-DR+) (Fig. 4C) and
activated T-cells (CD45+/CD56+/CD3−) (Fig. 4D) the same
findings can be reported, all with a p value of b 0.001.
3.3. Infant birth weight
Birth weight of the infant has been shown to impact on many of
the UCB cellular sub-populations. The babies in the study had
birth weights ranging from 2.585 kg to 4.425 kg, with an
average of 3.571 kg (±SD 0.44). When looking at TNC (Fig. 5A)
the data illustrates that babies with the lowest birth weight
also exhibit the lowest TNC and vice versa. This correlation was
significant with a p value b 0.0001. Birth weight also impacted
on HSC concentrations (Fig. 5B), especially mid-stage HSC. As
the birth weight rises so too does the HSC concentration
(p b 0.001). An optimum birth weight of between 3.25 and
3.75 kg gives a maximum yield of dendritic cells with ex-
pression of MHC class II molecules (Lin1−/CD11c+/HLA-DR+)
(Fig. 5C). The final sub-population to be described in relation
to infant birth weight is the T-cell. Once again, as birth weight
rises so does T-cell concentration (p b 0.001).
3.4. Gestational stage at parturition
Length of pregnancy was also investigated, with the standard
being a 40 week (280 day) period. The average gestation
length was 274.12 days (± SD 6.1) or 39 weeks and 1 day. It
was found that babies born either early or late by only a

Figure 4 Effects of the mother’s obstetric history on UCB cell populations. A. The graph illustrates the correlation between UCB TNC
and the number of pregnancies a woman has had. The results show that with each additional pregnancy the TNC in the UCB is more
likely to decrease. The correlation between number of pregnancies is significant with a p value b 0.0001. B. Early stage HSC
concentration in relation to the mother’s obstetric history. The correlation between the overall HSC (CD45+dim/CD34−/CD133+)
concentration and obstetric history shows a significance and a p value b 0.0001. C. Dendritic cells with expression of MHC class II
molecules (Lin1−/CD11c+/HLA-DR+) are significantly reduced with each pregnancy (p b 0.001). D. The same can be seen for activated
T-cell (CD56+/CD3−/CD45+) concentrations (p b 0.001). n = 43.
738 C.P. McGuckin et al.

Figure 5 Effects of the newborn’s birth weight on UCB cell populations. The babies in this study had birth weights ranging from
2.585 kg to 4.425 kg, with an average birth weight of 3.571 kg. A. The baby with the smallest weight had the lowest UCB TNC. The
trend line in this graph shows that a baby’s birth weight does have a significant effect on the TNC (p b 0.0001), with TNC increasing with
the increasing weight of the baby. B. HSC concentration in relation to the baby’s birth weight. This graph illustrates a correlation
between birth weight and mid-stage HSC concentration, in that HSC concentration increases with increasing birth weight of the baby.
The correlation is significant with a p value b 0.0001. C. An optimum birth weight of between 3.25 and 3.75 kg gives an optimum yield
of dendritic cells with expression of MHC class II molecules (Lin1−/CD11c+/HLA-DR+). D. T-cell (CD45+/CD3+) concentration increases,
with positive correlation, to infant birth weight (p b 0.0001). n = 43.

couple weeks tend to have lower UCB TNC than babies born
closer to the standard 40 week pregnancy (Fig. 6A)
(p b 0.001). Another observation made in relation to gesta-
tional stage is that there appears to be a clustering of higher
TNC when the baby is born between 38 and 40 weeks. When
looking at T-cell populations (Fig. 6B) and late stage HSC
populations (Fig. 6C) it was found that longer gestation
periods resulted in higher concentrations of these cell types
(p b 0.001 for both). However, when looking at B-cell
populations (CD45+/CD19+) within UCB there seems to be
an optimum gestation length of between 38 and 40 weeks,
when the concentration reaches its peak.
3.5. Mother’s age at parturition
The age of the mother at delivery, was also examined in
relation to the UCB cell populations. The age range of the 43
women involved was from 18 to 42 years with an average age
of 30.75 years (± SD 8.67). Mother’s age has a significant
impact upon late stage HSC populations (p b 0.0001). As age
increases, the HSC concentration is greatly reduced (Fig. 7A).
The same can be reported for the concentration of regulatory
T-cells (CD45+/CD4+/CD3+) (Fig. 7B) and indeed all lympho-
cytes (CD45+) (Fig. 7C) in UCB (p b 0.001). However, when
looking at the effects of mother’s age on UCB TNC, (Fig. 7D)
our data shows that mothers under the age of 20 and over the
age of 37 tend to have babies with lower TNC than mothers
that lie within that age range (p b 0.001).
4. Discussion
UCB stem cells have gained notoriety in both clinical and
research settings; however further work needs to be carried
out in order to maximise their potential. In the clinic, UCB
has become an alternative source of stem cells over BM and
PB. Presently, the recommended TNC content for UCB
transplantation is a minimum of 2 × 107/kg for adults and
3.7 × 107/kg for children [13]. Thus, it has also become
increasingly important to determine the best selection
processes for donors of UCB (to improve quality) and
storage of UCB units (to prevent storage of ineffective
blood units) based on obstetric factors that can influence
cell numbers.
It has previously been established that various obstetric
factors correlate not only with the UCB TNC, but also with
many UCB cell sub-type concentrations. Our study found that
UCB TNC content is affected by four of the five physiological
parameters that we examined. The newborn’s weight and
gestational stage are positively correlated with increasing
TNC concentration, which supports data found in previous
studies [12,20–22]. Mother’s age and obstetric history are
inversely proportional to TNC, evidence which is also
Cord blood revelations—The importance of being a first born girl, big, on time and to a young mother! 739

Figure 6 Effects of infant gestational stage on UCB cell populations. A. UCB TNC shows a positive correlation with gestational stage
at due date. The results demonstrate that babies born earlier than 37 weeks and later than 41 weeks show a reduced TNC. The
correlation between gestational stage and UCB TNC was significant with a p value b 0.0001. B. T-cell (CD45+/CD3+) concentration
increases, with positive correlation, to infant gestational stage at parturition (p b 0.0001). C. A correlation is observed between
gestational stage and late stage HSC (CD45+/CD34+/CD133−) concentration (p b 0.0001). The trend line shows that with increasing
gestational stage HSC concentration also increases. D. B-cell (CD45+/CD19+) concentration reaches an optimum level between 38 and
40 weeks. n = 43.

supported by previous studies [20,22,23]. The only parameter
investigated by our group that was not found to have any
correlation with UCB TNC concentration was gender, although
some studies show that females have higher TNC counts than
males [12]. Four of the five physiological parameters were
statistically significant with p values b 0.0001.
HSC (CD45+/CD34+) concentrations and HSC sub-types
were positively correlated with the baby’s weight and
gestational stage both of which support previous studies
[12,13,24]. HSC concentrations and sub-types were also
found to be inversely proportional to the number of parities
as well as mother’s age [23]. The only parameter to not show
a significant correlation was gender despite a trend showing
that baby girls have a higher HSC concentration than baby
boys.
These results for both TNC and HSC concentrations
suggest that the most valuable UCB units for banking and
thus transplantation come from full term, bigger babies who
are born to younger mothers with few previous pregnancies.
This could also affect immunity of babies, lower TNC pertains
to fewer lymphocytes.
Another cell type examined was the T-cells, which are
involved in cell-mediated immunity. There are many subsets
of T-cells and those examined in this study include activated
T-cells, those that have been stimulated by antigens and
have the function of both T-helper and T-cytotoxic cells, and
regulatory T-cells, those that are involved in immunological
tolerance by stopping T-cell mediated immunity at the end of
an immune response and suppressing auto-immune reactions
[25]. The most interesting effect on T-cells was shown by
obstetric history. Increased gravidity is inversely propor-
tional to T-cell concentration. Again this could have an
impact on the immunity of infants born late into larger
families. Mother’s age also negatively impacts T-cell con-
centration, and indeed all UCB lymphocytes, with older
mothers producing offspring with lower T-cell concentration.
However longer gestation and higher birth weights equate to
higher T-cell concentrations supporting the view that babies
who are born at full term are at lower risk than those born
prematurely [26].
Another UCB cellular sub-type examined, are B-cells.
These cells are concerned with the humoral immune
response and when activated in the presence foreign antigen
will make antibodies. However this process is complex and
assistance is required from T-helper cells [27]. B-cell
concentrations may be influenced by lower concentrations
of T-cells and consequently would also impact upon the
infant’s immunity.
Dendritic cells (DC) are professional antigen presenting
cells [28] and are of paramount importance in the immune
response. Both birth weight and obstetric history have high
impact on the levels of DC concentration found in UCB. The
740 C.P. McGuckin et al.

Figure 7 Effects of mother’s age on UCB cell populations. The age range of the 43 women involved in the study was from 18 to
42 years. A. This graph illustrates that with increasing age late stage HSC (CD45+/CD34+/CD133−) concentration decreases drastically,
with a p value b 0.0001. B. Regulatory T-cell (CD3+/CD4+/CD45+) concentration decreases significantly as the age of the mother
increases (p b 0.001). C. The same can be reported for lymphocytes (CD45+) in general within UCB. This negative correlation is
significant with a p value of b 0.001. D. Mother’s age does have an effect on the TNC and is demonstrated by the fact that a lower TNC
was observed for younger and older mothers. The correlation is significant with a p value b 0.0001. n = 43.

fact that a lower birth weight means lower DC concentra-
tions is of great concern. Premature babies therefore are
likely to have more problems with immunity than their full-
term counterparts. Again babies born to mothers with more
previous children are likely to suffer the same fate.
In the future it would be of interest to examine other
factors which have been shown to affect TNC and UCB
cellular sub-type concentrations such as placental weight,
which has been suggested to positively correlate with TNC
[11] and birth route. Vaginal deliveries have been suggested
to provide a greater TNC [23]. However results contrary to
this have also been shown [29]. Ethnic origin may also be an
interesting factor to examine as some previous work suggests
that it has no effect on TNC [22] but other work suggests that
Caucasian births deliver a higher number of T-helper (CD34+/
CD4+) cells [15]. However the main aim is to increase cohort
size in order to further validate the results we have already
found and to ascertain whether the gender-related trend is
significant. Results presented in this study demonstrated the
importance of characterising cell components within human
UCB samples prior to processing and cryo-preservation.
Acknowledgements
With thanks to the staff at Newcastle’s Royal Victoria
Infirmary Women’s Directorate and Maternity Unit and to
Newcastle University’s Stem Cell Laboratory team. The au-
thors are grateful to BioE Inc. (St Paul, MN, USA) for partly
funding this research study.
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