Page 78 - Human Umbilical Cord Mesenchymal Stem Cells

1478 R. Friedman et al.

BM-MSCs; they adhere to plastic, express characteris- sion in 20% FBS/RPMI 1640 (Cambrex, Walkersville,
tic surface markers, and can differentiate into cells MD) supplemented with antibiotics (penicillin 100 mg/
of mesenchymal origin, including bone, cartilage, mL, streptomycin 10 mg/mL, amphotericin B 250 mg/
adipose tissue, and neuronal cells [13-15]. Reports to mL, [all from Gibco, CA] and ciprofloxacin 10 mg/mL,
date have focused on obtaining those cells after culture [Mediatech, Herdon, VA]). Cultures were maintained

expansion from a segment of the umbilical cord [14- at 37 C in a humidified atmosphere containing 5%
16]. However, culture expansion has a disadvantage, CO 2 , with a change of 20% FBS/RPMI 1640 every
in that the cells cannot be frozen on the same day as 3-4 days. When colonies of fibroblast-like cells ap-
UCB cells arrive in the laboratory, and there is the in- peared and the wells reached 80% confluence, the cul-
creased risk of contamination with any culture manip- tures were rinsed with PBS, harvested with 0.05%
ulation. Thus, one objective of this study was to trypsin-EDTA (Gibco), and transferred into a 75-
2
develop a rapid same-day procedure for preparing cm flask for further expansion. Cell growth also was
and freezing UC-MSCs that would be suitable for clin- tested without FBS, using only RPMI 1640 or
ical application. X-Vivo 10 (Cambrex). For cryopreservation of the
Because previous studies have suggested that co- UC-MSCs, autologous UC plasma was obtained by
transplantation of BM-MSCs with hematopoietic centrifuging a UCB sample at 1700 rpm for 15 minutes
stem cells can accelerate hematopoietic engraftment at room temperature. Human serum (HS) was ob-
after transplantation [17,18], we hypothesized that tained from Cambrex. All sera were heat activated.
a similar effect could be accomplished when UC- Human BM-MSCs were isolated from BM aspi-
MSCs were used to support UCB transplantation. rates of healthy volunteers, after informed consent
UCB is a viable source of hematopoietic stem cells had been obtained. After Ficoll separation, the mono-
for treating hematologic diseases in both children nuclear cells (MNCs) were plated at a density of 10 6
2
and adults without a matched donor [19-21]. It has sev- cells/cm in 20% FBS/RPMI 1640. The culture ex-
eral advantages over matched unrelated donor marrow pansion protocol was the same as described for the
or peripheral blood stem cell transplantation: UCB UC-MSCs using 20% FBS/RPMI 1640.
units are more readily available, allow for some HLA UC-MSCs were stained with fluorescein isothio-
mismatches between patient and donor, and have cyanate (FITC)-conjugated antibodies against the fol-
a lower incidence of GVHD [22,23]. However, the lowing surface markers: CD3, CD14, CD19, CD34,
currently stored units will only allow transplantation CD45, CD49b, CD80, and HLA-class I (all obtained
of \10% of adults with an average weight of 70 kg from BD Pharmingen, San Diego, CA). Unconjugated
when 1 UCB unit is used [24]. Attempts at expanding Anti-3G5 and anti-STRO-1 were obtained from R&D
UCB ex vivo have yielded mixed results, with mostly Systems (Minneapolis, MN) and detected with second-
committed progenitors being expanded that are not ary anti-IgG FITC-conjugated antibody from Caltag
responsible for long-term engraftment [25]. (Burlingame, CA). Phycoerythrin (PE)-conjugated
Using standard transfection protocols, we also antibodies were used to identify the following surface
were able to show that UC-MSCs are amenable to markers: CD7, CD29, CD33, CD44, CD73, CD86,
gene transfection. This may be relevant to further en- and CD166. PE-Cy5–conjugated antibodies were
hance hematopoietic and immune cell engraftment used for CD58, CD117, and HLA-DR. Anti-CD40
and ex vivo expansion protocols in which UC-MSCs and anti-CD49d were conjugated with APC (all
may be used as feeders. antibodies obtained from BD Pharmingen). The PE-
conjugated anti-CD105 was from R&D Systems, and
METHODS the anti-CD133 was from Miltenyi (Auburn, CA).
Mouse isotypic antibodies (from BD Pharmingen)
Isolation and Culture
served as controls. Stained cells were analyzed by
Umbilical cords and cord blood were processed flow cytometry with a CyAn flow cytometer (Dako
within 24 hours after vaginal delivery or cesarean Colorado, Ft. Collins, CO).
section. The protocol was approved by Tufts-New
England Medical Center Institutional Review Board.
The cords were delivered in a sterilized jar to the lab- Cytokine Production of UC-MSCs and BM-MSCs
oratory and kept at room temperature if they could not To assess cytokine production by UC-MSCs and
4
be prepared immediately. After washing in phosphate- BM-MSCs, 10 cells/well of each cell type were seeded
buffered saline (PBS) to remove any contaminating into 6 well tissue culture plates (BD Biosciences,
blood, the cord was cut into small pieces (0.5-1 cm), Franklin Lakes, NJ) and grown to confluence in 20%
and the vessels were removed to avoid endothelial FBS/RPMI 1640 medium for 7 days with no medium
cell contamination. The pieces were either minced change. The supernatant was removed and new me-
and cryopreserved, as described in the Results section, dium was supplied to the wells for 24 hours, after
or placed directly into 6 well plates for culture expan- which the supernatant was collected and sent to Pierce