Page 79 - Human Umbilical Cord Mesenchymal Stem Cells

Umbilical Cord MSC to Support Cell Transplantation 1479

Biotech (Woburn, MA) to be analyzed by standard CA). GFP mRNA was synthesized in vitro from a line-
enzyme-linked immunosorbent assay (ELISA) for arized GFP-pXT7 plasmid template (with the pXT7
the following cytokines/growth factors: granulocyte a gift from Dr S. Sokol), using the T7 mMessage mMa-
colony-stimulating factor (G-CSF), granulocyte chine kit (Ambion, Austin, TX) according to the man-
macrophage colony-stimulating factor (GM-CSF), ufacturer’s instructions. UC-MSCs were trypsinized
thrombopoietin (TPO), vascular endothelial growth and resuspended in MEM medium (Gibco, Carlsbad,
6
factor (VEGF), stromal cell derived factor-1 (SDF)- CA) at a concentration of 2 10 cells/mL. Electropo-
1b, transforming growth factor (TGF)-a, TGF-b, hu- ration was performed on a Genepulser II (Bio Rad,
5
man growth factor (HGF), interferon (IFN)-a, IFN-g, Hercules, CA) on 4 10 cells (200 mL) per 4-mm
tumor necrosis factor (TNF)-a, leukemia inhibitory cuvette using different voltages (150 and 300 V) and
factor (LIF), interleukin (IL)-1a, IL-2, IL-6, IL-7, capacitances (150 and 300 mF). DNA (75 mg/mL) or
IL-8, IL-11, IL-12p40, IL-15, and IL-18. Media mRNA (25 mg/mL) was added directly to the cell
with no MSCs were tested as controls. suspension and incubated for 2 minutes at room tem-
perature before electroporation. After transfection,
Hematopoietic Colony Formation Assay
cells were transferred to 6 well plates with 20% FBS/

UC-MSCs were plated at a concentration of 2 RPMI and allowed to grow for 40 hours at 37 C.
5
10 cells, grown to confluence in 35-mm culture plates, Expression of GFP was analyzed by flow cytometry
1
and irradiated with 3200 cGy. UCB CD34 cells were of trypsinized cells stained with propidium iodide
selected using the Miltenyi Biotec MiniMACS system (10 mg/mL in PBS) before analysis.
3
and suspended at a concentration of 1 10 cells/mL
in MethoCult H4434 (StemCell Technologies, Van- Statistical Analysis
couver, Canada). The cells were then plated on top Data are presented as mean 6 standard error of the
of the UC-MSCs containing culture plates as well as mean unless otherwise noted. Statistical analyses using
on plates cultured without MSCs, which served as con- SPSS 11.5 software (SPSS, Chicago, IL) were per-
trols. The numbers of CFU-GEMM, BFU-E, and formed with the Student t test. All p values \.05
CFU-GM colonies were counted on day 14. were considered statistically significant.

Co-Transplantation of NOD/SCID Mice with
UCB Cells and UC-MSCs RESULTS
All experiments and procedures in animals were Rapid Preparation of UC-MSCs for
performed in compliance with the regulations for ani- Cryopreservation under GMP Conditions
mal experimentation at Tufts-New England Medical One objective of this study was to show that UC-
Center. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD/ MSCs could be prepared and frozen without previous
SCID gcnull) mice were purchased from Jackson Lab- culture expansion. To further avoid storage in animal
oratories (Bar Harbor, ME) and were maintained in or human allogeneic serum, we tested whether a small
sterile microisolator cages under specific pathogen- piece of cord (0.5-1 cm) could be minced and immedi-
free conditions and provided with irradiated food. ately frozen in autologous cord plasma and still maintain
Four groups of 8 mice each (age 8-10 weeks) were sub- all of the characteristics of fresh and culture-expanded
lethally irradiated with 350 cGy and underwent trans- UC-MSCs. Blood vessels were removed from the
plantation with the following cells from the same cord cord section, and the remaining Wharton’s jelly was
3
6
donor: 10 umbilical cord MNCs alone or together cut into 1- to 2-mm fragments using sterile scissors.
4
6
with 10 UC-MSCs or 10 CD34 selected cells alone The tissue particles were further minced and then
6
or together with 10 UC-MSCs. Cells were suspended mixed with 10 % DMSO and heat-inactivated autolo-
in 250 mL of PBS and injected slowly into a lateral tail gous cord plasma. The minced pieces were transferred
vein. The mice were sacrificed 6-8 weeks after trans- into a 5-mL VueLife bag (AFC, Gaithersburg, MD) or

plantation. Peripheral blood samples were obtained cryovials and stored at -80 C until further use. To test
by cardiac puncture, and BM was harvested by flushing whether UC-MSCs can be expanded from the frozen
femurs and tibias with PBS. Flow cytometry for the cord pieces, the thawed, minced microparticles were
HLA CD45 was used to document engraftment with placed into 6 well plates in the presence of 5 different
FITC-conjugated antibody to human CD45 (BD serum conditions (10% or 20% FBS, 10% or 20% HS,
Pharmingen) and PE-conjugated antibody to murine and autologous cord plasma). Serum was sup-
CD45 (eBioscience, San Diego, CA). plemented with either X-Vivo10 or RPMI 1640. The
viability of thawed UC-MSCs was consistently
Transfection of UC-MSCs with Plasmid DNA
.90% by trypan blue staining. The medium was
or mRNA for GFP
changed every 3-4 days. UC-MSCs plated with FBS
DNA electroporation was performed using the (10% or 20%) became readily adherent and displayed
pEGFP-C1 plasmid (Clontech, Mountain View, the fastest growth kinetics (Figure 1A). FBS at 20%