Biology of Blood and Marrow Transplantation
Volume 13, Issue 12 , Pages 1477-1486, December 2007

Umbilical Cord Mesenchymal Stem Cells: Adjuvants for Human Cell Transplantation

Tufts-New England Medical Center, Molecular Oncology Research Institute, Boston, Massachusetts

Received 3 July 2007; accepted 30 August 2007.

Article Outline

Abstract 

The Wharton's jelly of the umbilical cord is rich in mesenchymal stem cells (UC-MSCs) that fulfill the criteria for MSCs. Here we describe a novel, simple method of obtaining and cryopreserving UC-MSCs by extracting the Wharton's jelly from a small piece of cord, followed by mincing the tissue and cryopreserving it in autologous cord plasma to prevent exposure to allogeneic or animal serum. This direct freezing of cord microparticles without previous culture expansion allows the processing and freezing of umbilical cord blood (UCB) and UC-MSCs from the same individual on the same day on arrival in the laboratory. UC-MSCs produce significant concentrations of hematopoietic growth factors in culture and augment hematopoietic colony formation when co-cultured with UCB mononuclear cells. Mice undergoing transplantation with limited numbers of human UCB cells or CD34+ selected cells demonstrated augmented engraftment when UC-MSCs were co-transplanted. We also explored whether UC-MSCs could be further manipulated by transfection with plasmid-based vectors. Electroporation was used to introduce cDNA and mRNA constructs for GFP into the UC-MSCs. Transfection efficiency was 31% for cDNA and 90% for mRNA. These data show that UC-MSCs represent a reliable, easily accessible, noncontroversial source of MSCs. They can be prepared and cryopreserved under good manufacturing practices (GMP) conditions and are able to enhance human hematopoietic engraftment in SCID mice. Considering their cytokine production and their ability to be easily transfected with plasmid-based vectors, these cells should have broad applicability in human cell–based therapies.

Key Words: Cord blood transplantation, Mesenchymal stem cell, Natural killer cell

 

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Introduction 

Mesenchymal stem cells (MSCs) are being increasingly developed for indications in the growing field of regenerative medicine and also for their ability to modulate the immune response 1, 2. Recent clinical trials have suggested that bone marrow–derived MSCs (BM-MSCs) can aid children born with osteogenesis imperfecta [3], improve cardiac function after myocardial infarction [4], and treat acute graft-versus-host disease (aGVHD) after bone marrow (BM) transplantation 5, 6. Given that harvesting of BM-MSCs involves a painful, invasive procedure, alternative sources of MSCs may prove more useful. Recently, extraction and characterization of MSCs derived from fetal tissues, such as the placenta and the umbilical cord, as well as from umbilical cord blood (UCB), have been described 7, 8, 9, 10, 11.

During embryogenesis, cells from the hematopoietic progenitor tissue of the fetus, the aortamesogonad region, migrate through the developing cord. It is believed that some of these early cells are trapped there and contribute to the cellular composition of the cord [12]. Inside the cord is a jelly-like matrix material that protects the cord arteries and the vein, initially described by Thomas Wharton in 1656 (“Wharton's jelly”). Termed umbilical cord MSCs (UC-MSCs), these cells have many of the same characteristics as BM-MSCs; they adhere to plastic, express characteristic surface markers, and can differentiate into cells of mesenchymal origin, including bone, cartilage, adipose tissue, and neuronal cells 13, 14, 15. Reports to date have focused on obtaining those cells after culture expansion from a segment of the umbilical cord 14, 15, 16. However, culture expansion has a disadvantage, in that the cells cannot be frozen on the same day as UCB cells arrive in the laboratory, and there is the increased risk of contamination with any culture manipulation. Thus, one objective of this study was to develop a rapid same-day procedure for preparing and freezing UC-MSCs that would be suitable for clinical application.

Because previous studies have suggested that co-transplantation of BM-MSCs with hematopoietic stem cells can accelerate hematopoietic engraftment after transplantation 17, 18, we hypothesized that a similar effect could be accomplished when UC-MSCs were used to support UCB transplantation. UCB is a viable source of hematopoietic stem cells for treating hematologic diseases in both children and adults without a matched donor 19, 20, 21. It has several advantages over matched unrelated donor marrow or peripheral blood stem cell transplantation: UCB units are more readily available, allow for some HLA mismatches between patient and donor, and have a lower incidence of GVHD 22, 23. However, the currently stored units will only allow transplantation of <10% of adults with an average weight of 70 kg when 1 UCB unit is used [24]. Attempts at expanding UCB ex vivo have yielded mixed results, with mostly committed progenitors being expanded that are not responsible for long-term engraftment [25].

Using standard transfection protocols, we also were able to show that UC-MSCs are amenable to gene transfection. This may be relevant to further enhance hematopoietic and immune cell engraftment and ex vivo expansion protocols in which UC-MSCs may be used as feeders.

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Methods 

Isolation and Culture 

Umbilical cords and cord blood were processed within 24 hours after vaginal delivery or cesarean section. The protocol was approved by Tufts-New England Medical Center Institutional Review Board. The cords were delivered in a sterilized jar to the laboratory and kept at room temperature if they could not be prepared immediately. After washing in phosphate-buffered saline (PBS) to remove any contaminating blood, the cord was cut into small pieces (0.5-1 cm), and the vessels were removed to avoid endothelial cell contamination. The pieces were either minced and cryopreserved, as described in the Results section, or placed directly into 6 well plates for culture expansion in 20% FBS/RPMI 1640 (Cambrex, Walkersville, MD) supplemented with antibiotics (penicillin 100 μg/mL, streptomycin 10 μg/mL, amphotericin B 250 μg/mL, [all from Gibco, CA] and ciprofloxacin 10 μg/mL, [Mediatech, Herdon, VA]). Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2, with a change of 20% FBS/RPMI 1640 every 3-4 days. When colonies of fibroblast-like cells appeared and the wells reached 80% confluence, the cultures were rinsed with PBS, harvested with 0.05% trypsin-EDTA (Gibco), and transferred into a 75-cm2 flask for further expansion. Cell growth also was tested without FBS, using only RPMI 1640 or X-Vivo 10 (Cambrex). For cryopreservation of the UC-MSCs, autologous UC plasma was obtained by centrifuging a UCB sample at 1700 rpm for 15 minutes at room temperature. Human serum (HS) was obtained from Cambrex. All sera were heat activated.

Human BM-MSCs were isolated from BM aspirates of healthy volunteers, after informed consent had been obtained. After Ficoll separation, the mononuclear cells (MNCs) were plated at a density of 106 cells/cm2 in 20% FBS/RPMI 1640. The culture expansion protocol was the same as described for the UC-MSCs using 20% FBS/RPMI 1640.

UC-MSCs were stained with fluorescein isothiocyanate (FITC)-conjugated antibodies against the following surface markers: CD3, CD14, CD19, CD34, CD45, CD49b, CD80, and HLA-class I (all obtained from BD Pharmingen, San Diego, CA). Unconjugated Anti-3G5 and anti-STRO-1 were obtained from R&D Systems (Minneapolis, MN) and detected with secondary anti-IgG FITC-conjugated antibody from Caltag (Burlingame, CA). Phycoerythrin (PE)-conjugated antibodies were used to identify the following surface markers: CD7, CD29, CD33, CD44, CD73, CD86, and CD166. PE-Cy5–conjugated antibodies were used for CD58, CD117, and HLA-DR. Anti-CD40 and anti-CD49d were conjugated with APC (all antibodies obtained from BD Pharmingen). The PE-conjugated anti-CD105 was from R&D Systems, and the anti-CD133 was from Miltenyi (Auburn, CA). Mouse isotypic antibodies (from BD Pharmingen) served as controls. Stained cells were analyzed by flow cytometry with a CyAn flow cytometer (Dako Colorado, Ft. Collins, CO).

Cytokine Production of UC-MSCs and BM-MSCs 

To assess cytokine production by UC-MSCs and BM-MSCs, 104 cells/well of each cell type were seeded into 6 well tissue culture plates (BD Biosciences, Franklin Lakes, NJ) and grown to confluence in 20% FBS/RPMI 1640 medium for 7 days with no medium change. The supernatant was removed and new medium was supplied to the wells for 24 hours, after which the supernatant was collected and sent to Pierce Biotech (Woburn, MA) to be analyzed by standard enzyme-linked immunosorbent assay (ELISA) for the following cytokines/growth factors: granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), thrombopoietin (TPO), vascular endothelial growth factor (VEGF), stromal cell derived factor-1 (SDF)-1β, transforming growth factor (TGF)-α, TGF-β, human growth factor (HGF), interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, leukemia inhibitory factor (LIF), interleukin (IL)-1α, IL-2, IL-6, IL-7, IL-8, IL-11, IL-12p40, IL-15, and IL-18. Media with no MSCs were tested as controls.

Hematopoietic Colony Formation Assay 

UC-MSCs were plated at a concentration of 2 × 105 cells, grown to confluence in 35-mm culture plates, and irradiated with 3200 cGy. UCB CD34+ cells were selected using the Miltenyi Biotec MiniMACS system and suspended at a concentration of 1 ×103 cells/mL in MethoCult H4434 (StemCell Technologies, Vancouver, Canada). The cells were then plated on top of the UC-MSCs containing culture plates as well as on plates cultured without MSCs, which served as controls. The numbers of CFU-GEMM, BFU-E, and CFU-GM colonies were counted on day 14.

Co-Transplantation of NOD/SCID Mice with UCB Cells and UC-MSCs 

All experiments and procedures in animals were performed in compliance with the regulations for animal experimentation at Tufts-New England Medical Center. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD/SCID γcnull) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and were maintained in sterile microisolator cages under specific pathogen-free conditions and provided with irradiated food. Four groups of 8 mice each (age 8-10 weeks) were sublethally irradiated with 350 cGy and underwent transplantation with the following cells from the same cord donor: 106 umbilical cord MNCs alone or together with 106 UC-MSCs or 104 CD34 selected cells alone or together with 106 UC-MSCs. Cells were suspended in 250 μL of PBS and injected slowly into a lateral tail vein. The mice were sacrificed 6-8 weeks after transplantation. Peripheral blood samples were obtained by cardiac puncture, and BM was harvested by flushing femurs and tibias with PBS. Flow cytometry for the HLA CD45 was used to document engraftment with FITC-conjugated antibody to human CD45 (BD Pharmingen) and PE-conjugated antibody to murine CD45 (eBioscience, San Diego, CA).

Transfection of UC-MSCs with Plasmid DNA or mRNA for GFP 

DNA electroporation was performed using the pEGFP-C1 plasmid (Clontech, Mountain View, CA). GFP mRNA was synthesized in vitro from a linearized GFP-pXT7 plasmid template (with the pXT7 a gift from Dr S. Sokol), using the T7 mMessage mMachine kit (Ambion, Austin, TX) according to the manufacturer's instructions. UC-MSCs were trypsinized and resuspended in MEM medium (Gibco, Carlsbad, CA) at a concentration of 2 × 106 cells/mL. Electroporation was performed on a Genepulser II (Bio Rad, Hercules, CA) on 4 × 105 cells (200 μL) per 4-mm cuvette using different voltages (150 and 300 V) and capacitances (150 and 300 μF). DNA (75 μg/mL) or mRNA (25 μg/mL) was added directly to the cell suspension and incubated for 2 minutes at room temperature before electroporation. After transfection, cells were transferred to 6 well plates with 20% FBS/RPMI and allowed to grow for 40 hours at 37°C. Expression of GFP was analyzed by flow cytometry of trypsinized cells stained with propidium iodide (10 μg/mL in PBS) before analysis.

Statistical Analysis 

Data are presented as mean ± standard error of the mean unless otherwise noted. Statistical analyses using SPSS 11.5 software (SPSS, Chicago, IL) were performed with the Student t test. All p values <.05 were considered statistically significant.

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Results 

Rapid Preparation of UC-MSCs for Cryopreservation under GMP Conditions 

One objective of this study was to show that UC-MSCs could be prepared and frozen without previous culture expansion. To further avoid storage in animal or human allogeneic serum, we tested whether a small piece of cord (0.5-1 cm) could be minced and immediately frozen in autologous cord plasma and still maintain all of the characteristics of fresh and culture-expanded UC-MSCs. Blood vessels were removed from the cord section, and the remaining Wharton's jelly was cut into 1- to 2-mm3 fragments using sterile scissors. The tissue particles were further minced and then mixed with 10 % DMSO and heat-inactivated autologous cord plasma. The minced pieces were transferred into a 5-mL VueLife bag (AFC, Gaithersburg, MD) or cryovials and stored at -80°C until further use. To test whether UC-MSCs can be expanded from the frozen cord pieces, the thawed, minced microparticles were placed into 6 well plates in the presence of 5 different serum conditions (10% or 20% FBS, 10% or 20% HS, and autologous cord plasma). Serum was supplemented with either X-Vivo10 or RPMI 1640. The viability of thawed UC-MSCs was consistently >90% by trypan blue staining. The medium was changed every 3-4 days. UC-MSCs plated with FBS (10% or 20%) became readily adherent and displayed the fastest growth kinetics (Figure 1A). FBS at 20% promoted growth significantly better than FBS at 10%. Significantly slower growth was observed in cells cultured in HS. The cell morphology also differed, in that UC-MSCs in HS grew in clumps with fewer cells spreading out on plastic (Figure 1B). Cord plasma did not support the expansion of UC-MSCs. No significant differences were observed when either X-Vivo10 or RPMI 1640 was used as the base medium. Notably, neither X-Vivo10 nor RPMI 1640 by itself supported the expansion of UC-MSCs in culture. There was also no difference as to whether cells were initially cryopreserved in either VueLife bags or cyrovials (data not shown). The doubling time was calculated for UC-MSCs derived from 6 UC and expanded in FBS 20%/RPMI 1640. The mean doubling time was 2.26 days (range, 1.6-3.3) for up to 7 passages (data not shown).

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  • Figure 1 

    A, Expansion of UC-MSCs in various culture media. Fresh cord pieces were minced and those particles were cryopreserved in 10% DMSO and autologous cord plasma to avoid exposure of UC-MSCs to allogeneic media. After thawing, UC-MSCs were expanded in 20% FBS, 10% FBS, 20% HS, 10% HS, or autologous cord plasma. The number of expanded cells is plotted on the y-axis. All sera were heat-inactivated and supplemented with either RPMI 1640 or X-Vivo10. FBS at 20% resulted in the best expansion. No difference was seen for RPMI 1640 or X-Vivo10 as base media. Results from 1 of 3 representative experiments are presented. B, Microscopic view of UC-MSCs cultured in either 20% FBS/RPMI 1640 or 20% HS/RPMI 1640 appearance (original magnification × 40). In HS, the MSCs tend to form clumps, with less UC-MSCs becoming adherent. Spreading of the clumps can be observed when plucked and replated in 20% FBS.

To determine whether there is an area of the cord that is particularly rich in UC-MSCs, representative samples from different areas of the entire length of the cord were obtained, and cells were cultured for 7, 14, and 21 days. No significant differences were noted with respect to expansion kinetics among UC-MSCs obtained from the areas adjacent to the placenta, middle and distal, or adjacent to the umbilicus (data not shown).

Antigen Expression on UC-MSCs 

Table 1 compares the surface antigen expression of UC-MSCs and BM-MSCs. UC-MSCs cultured in 20% FBS/RPMI expressed the following surface antigens to various extents: CD29, CD44, CD49b, CD58, CD73, CD105, CD166, and HLA-ABC. UC-MSCs stained negative for the following surface antigens: CD3, CD7, CD14, CD19, CD33, CD34, CD40, CD45, CD49d, CD80, CD86, CD117, CD133, 3G5, STRO-1, and HLA-DR. Cells cultured in 10% or 20% HS instead of FBS demonstrated the same antigen expression profile (data not shown).

Table 1. Percentages of UC-MSCs and BM-MSCs expressing surface markers as determined by flow cytometry
MarkerUC-MSCBM-MSC
CD3--
CD7--
CD14--
CD19--
CD29++++++
CD33--
CD34--
CD40--
CD44++++++
CD45--
CD49b++-
CD49d--
CD58++++++
CD73+++++++
CD80--
CD86--
CD105++++++++
CD117--
CD133--
CD166++++++
HLA ABC+++
HLA DR--
3G5--
STRO-1--

The expression levels are as follows: + = 0%-25%; ++ = 26%-50%; +++ = 51%-75%; ++++ = 76%-100%.

The data present the average of 4 independent experiments.

Cytokine Production of UC-MSCs and BM-MSCs 

Table 2 compares the concentrations of cytokines produced by UC-MSCs and BM-MSCs. UC-MSCs secrete significantly higher concentrations of G-CSF, GM-CSF, HGF, LIF, IL-1α, IL-6, IL-8, and IL-11 compared with BM-MSCs, whereas BM-MSCs produce more VEGF and SDF-1β than UC-MSCs. Production of IL-2, IL-7, IL-12p40, IL-15, IL-18, IFN-α, IFN-γ, and TNF-α was negligible for both UC-MSCs and BM-MSCs.

Table 2. Cytokine production by FBS, UC-MSCs, and BM-UCMs, as measured by ELISA
FBSUC-MSCsBM-MSCsP
GCSF<1.61845.7 ± 881.01.1 ± 0.3.007
GM-CSF<11.7118.9 ± 19.68.3 ± 3.3.023
TPO83.2165.7 ± 4.5160.2 ± 2.6NS
VEGF31.5151.4 ± 4.72762.1 ± 99.0<.001
SDF1β<6.3408.9 ± 53.12348.2 ± 177.6<.001
TGFβ7267.511079.9 ± 584.69493.8 ± 30.8NS
HGF1.012596.8 ± 7101.2402.6 ± 203<.001
LIF17.1169.2 ± 34.958.5 ± 2.5.001
TNF-α<4.73.4 ± 1.14.9 ± 1.3NS
IFN-α0.14.0 ± 0.52.4 ± 0.2NS
IFN-γ<0.41.9 ± 0.30.6 ± 0.1NS
IL-1a<0.412.5 ± 3.870.5 ± 0.1.001
IL-2<0.4<0.4<0.4NS
IL-6<0.41571.0 ± 617.2704.0 ± 51.5.002
IL-70.97.72 ± 1.08.85 ± 0.8NS
IL-8<0.410765.3 ± 5382.6216.9 ± 26.6<.001
IL-11<21474.4 ± 625.371.8 ± 0.7.001
IL-12<0.4<0.4<0.4NS
IL-150.75.1 ± 0.35.0 ± 0.1NS
IL-180.820.8 ± 1.217.9 ± 0.1NS

All values represent the mean of 4 experiments ± standard error of the mean, in pg/mL of cytokine.

NS indicates not significant.

UC-MSCs Increase Hematopoietic Progenitor Cell Proliferation 

CD34+ cells from human UCB were suspended in Methocult and either plated on a monolayer of irradiated (3200 cGy) UC-MSCs grown to confluence or plated in a culture dish without a UC-MSC monolayer (Methocult only). Hematopoietic colony formation was assessed after 14 days of incubation. Significantly more CFU-GM colonies (184 ± 34 vs 94 ± 16; P = .017), but not BFU-E (126 ± 11 vs. 70 ± 20; P = .06) or CFU-GEMM (35 ± 4 vs 15 ± 3; P = .6) colonies were generated in the presence of the UC-MSC monolayer than without after 2 weeks of culture.

Co-Transplantation of NOD/SCID γcnull Mice with UCB and UC-MSCs 

Irradiated NOD/SCID γcnull mice were injected either with UCB cells (unfractionated MNCs) or selected CD34+ cells either with and without 106 UC-MSCs, extracted from the same UC as the UCB. BM and peripheral blood were harvested 6-8 weeks after transplantation and analyzed for human cell engraftment based on the number of human CD45+ cells found. As shown in Figure 2, mice undergoing transplantation with UCB cells alone demonstrated 1.9% human CD45+ cell engraftment in the BM and 5.9% human CD45+ cells in peripheral blood, whereas mice receiving the same number of UCB cells and 106 UC-MSCs had an average of 6.7% human CD45+ cells engrafted in the BM (P = .017) and 38.4% human CD45+ cells engrafted in the peripheral blood (P = .001). Similar results were observed when CD34+ selected cells were transplanted instead of unfractionated MNCs. CD34+ cells alone resulted in 4.5% of human cells in the BM and 2.7% in peripheral blood, whereas co-transplantation with UC-MSCs resulted in 10.1% human cells in BM and 2.1 % in blood. The difference for BM was significant (P = .001).

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  • Figure 2 

    Engraftment of human BM cells in NOD/SCID mice. The mice underwent transplantation (intravenous injection) either with UCB cells at 106 alone or together with 106 UC-MSCs (A) or with CD34-enriched UCB cells at 104 alone or together with 106 UC-MSCs (B). Human cell engraftment was assessed at 6-8 weeks according to the human CD45 marker. Co-transplanted UC-MSCs augmented engraftment for transplantation of unfractionated UCB cells as well as CD34 selected hematopoietic precursors.

In some mice, human MSCs, as evidenced by the expression of CD105+, were found in BM of mice co-transplanted with UC-MSCs but not in mice transplanted with UCB alone. This was not seen consistently in all mice that underwent transplantation, however. Furthermore, some mice that were co-transplanted with UCB and UC-MSCs showed a positive signal for CD41 in blood, which identifies the GPIIa/IIIb protein on platelets (results not shown).

Transfection of UC-MSCs with cDNA and mRNA 

UC-MSCs have potentially broad clinical applicability, and it is relevant to show that they can be manipulated to express certain surface molecules involved in homing to target organs to produce chemokines and cytokines to support hematopoiesis or to mediate antitumor effects. We compared transfection of UC-MSCs with a plasmid-based cDNA construct and transfection with mRNA. Results presented in Figure 3 show that UC-MSC can be readily transfected with the GFP reporter gene. The transfection efficiency for cDNA was 30.9% and that for mRNA was 89.8%. Significant expression of GFP after mRNA transfection was maintained for at least 4 days.

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  • Figure 3 

    Transfection of UC-MSCs with the GFP reporter gene. Adherent cells were transfected using the BioRad electroporator with either cDNA or mRNA at different voltages and capacitance, as indicated. mRNA showed higher transfection efficiency than cDNA, with expression of the GFP gene maintained for several days.

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Discussion 

MSCs can be isolated from multiple sources and have different properties depending on the tissue origin and degree of differentiation. MSCs derived from human BM are in clinical trials to treat Crohn's disease, to treat GVHD after stem cell transplantation (SCT), or to support bone and cartilage regeneration. These cells are known to play a vital role in the hematopoietic niche and serve as structural elements and nurse cells for hematopoietic cells [26]. Based on these observations, BM-MSCs seem to be ideal candidates to support hematopoietic cell engraftment after stem cell transplantation, especially given the low hematopoietic stem cell dose often found in UCB, which frequently is the cause of delayed engraftment or graft failure in transplant recipients. As shown here, UC-MSCs are readily extracted from the Wharton's jelly of the UC and thus represent a readily accessible abundant source of MSCs to aid in UCB transplantation. They have been shown to differentiate into other tissues of mesenchymal origin, such as adipocytes, cartilage, bone, and nerve cells 14, 15, 16.

Our method of cryopreserving UC-MSCs from the fresh cord early after delivery with minimal manipulation and cryopreservation in autologous cord plasma avoids exposure of UC-MSCs to either animal serum or allogeneic HS. Both serum preparations are known to alter the gene profile and antigen expression of MSCs 27, 28. In addition, FBS or allogeneic HS potentially could be contaminated with viruses or prions, and preserving UC tissue in autologous cord plasma avoids those risks. Mincing and freezing the cord tissue in autologous cord serum is of practical importance from a UCB banking perspective, because this method avoids previous culture expansion of UC-MSCs. It allows for freezing of the UC-MSCs at the same time as the UCB arrives, and both cell types can be stored in the same dual-chamber bag. This facilitates the use of autologous UC-MSCs for any medical indication (beyond transplantation) at a later point. Our results also confirm that large numbers of UC-MSCs can be expanded in culture from a small piece of cord that has been finely minced and that the microparticles can be cryopreserved on arrival in the laboratory. When preparing the cord, special care was taken not to collect the perivascular cells in the cord (pericytes), which have different characteristics despite being MSCs [9]. Our cells were consistently negative for the 3G5 surface antigen, suggesting that our preparations were free of pericytes.

The optimum medium in which to expand UC-MSCs after thawing is 20% FBS in either RPMI 1640 or X-Vivo10. Inferior results were obtained with 10% FBS or HS, even at 20%. The cells did not expand in autologous cord plasma at all. The requirement of FBS for optimum expansion is not unique for UC-MSCs; human BM-MSCs also grow best in FBS [29]. Consequently, even the BM-MSCs used in current clinical trials were expanded in FBS. Recent data suggest though that serum obtained from platelet-rich plasma support MSC expansion [30]. Our results further demonstrate that UC-MSCs grow well in X-Vivo10 when used to supplement FBS. A drug master file exists with the FDA for X-Vivo10 for use in clinical trials.

UCB is a viable source of hematopoietic stem cells for transplantation in both children and adults undergoing treatment for hematologic malignancies and some nonmalignant disorders. However, the utility of UCB transplantation in adults is limited by the total cell dose contained in a single stored unit of UCB, and attempts of ex vivo expansion of UCB have shown that largely committed hematopoietic progenitors expand that are unable to provide long-term engraftment [25]. Transplantation of 2 or more umbilical cords has been found to shorten engraftment after transplantation to some degree [31]; however, questions remain about the long-term effects of transplanting multiple immune systems, in addition to the increased procurement costs.

Co-transplantation of hematopoietic stem cells and MSCs obtained from BM has demonstrated enhanced engraftment in NOD/SCID mice, especially when hematopoietic stem cells were given at suboptimal doses 16, 17, 32, 33. These encouraging results led to human trials that demonstrated that infusion of MSC in adults is safe and well tolerated; however, none of these trials demonstrated a conclusive engraftment advantage, likely because the number of hematopoietic stem cells transplanted from BM was already sufficiently high to allow timely engraftment 34, 35. We have shown that in a NOD/SCID mouse model, human UC-MSCs, when co-injected with human UCB cells, can accelerate human hematopoietic stem cell recovery when limited numbers of UCB cells or CD34 cells are injected. This improved engraftment may be explained by the ability of UC-MSCs to provide a matrix or stroma for engrafting stem cells. Although studies by Noort et al [17] indicated that intravenously administered MSCs are initially “trapped” in the lung and then possibly recirculate, our data show that small numbers of human UC-MSCs can be found in the BM, as demonstrated by the presence of human CD105+ cells in the marrow of some mice (data not shown). This assessment was done 6 weeks after infusion of UC-MSCs, which may have been too early to reflect a more robust engraftment of MSC. Muguruma et al [26] reported that significant human MSC engraftment was detected in the marrow of SCID mice at 6 months. We show here that UC-MSCs produce hematopoietic growth factors and the augmented human cell engraftment seen in SCID mice also could be facilitated by the production of those cytokines, released by co-injected UC-MSCs (eg, GM-CSF, G-CSF, IL-1, IL-8, IL-11). Of potential clinical interest is our preliminary observation of human platelets in the BM of SCID mice when they were co-transplanted with UC-MSCs.

The UCB cells and UC-MSCs in this study were injected intravenously, and it is possible that injection of both cell types directly into the BM cavity could accelerate engraftment to an even greater extent, because cells would not have to pass through the lung and other organs. Intra-BM transplantation has been explored in the murine system 36, 37.

Another clinical indication for UC-MSCs is their use as a feeder layer to expand hematopoietic stem cells, immune cells, or any other cell type. Studies using BM-MSCs have suggested that expansion of UCB cells was superior when they were co-cultured with MSCs [33]. We have shown that co-culture of UCB cells with UC-MSCs resulted in significantly more hematopoietic colonies than without UC-MSCs. We also have recently shown that UCB natural killer cells are effectively expanded when cultured with cytokines on a feeder layer of UC-MSCs [38].

To further develop UC-MSCs as “custom” feeders for expansion of human cells, we exploited their ability to be transfected with target genes, using GFP as a model gene. To avoid a retrovirus-based construct, we used a plasmid-based vector and electoporation. Cultured adherent UC-MSCs displayed excellent transfection efficiency both for cDNA and mRNA vectors, with expression of the marker gene maintained for at least 2 days. Not unexpectedly, transfection efficiency was greater with mRNA, because the transfected construct does not have to enter the nucleus. mRNA is not integrated into the genome and usually is degraded over time, simplifying the regulatory requirements. However, a disadvantage could be the time-limited expression of the mRNA. In addition to using such engineered UC-MSCs as feeder layers, they also could be used as carriers for anti-inflammatory or tumoricidal compounds, because MSCs have been shown to home to sites of inflammation and malignant histology 39, 40, 41. Their easy “transfectability” and low allogeneic potential because of low HLA class I expression could make these cells ideal candidates for this indication.

In conclusion, UC-MSCs hold significant promise in the emerging field of regenerative medicine and cellular therapy. We have described a novel method of obtaining UC-MSCs for rapid cryopreservation and showed that these cells can support engraftment of a limited number of human UCB hematopoietic stem cells in a NOD/SCID transplant model. Furthermore, we have shown that UC-MSCs can be readily transfected with a plasmid-based vector through electroporation. These characteristics make UC-MSC ideal platform candidates for a broad range of cell-based therapies.

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Acknowledgments 

This work was supported by a grant from the Russo Foundation through the Tufts School of Medicine. We thank Sandra Turcios for assisting in the preparation of UC-MSCs.

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PII: S1083-8791(07)00437-5

doi:10.1016/j.bbmt.2007.08.048

Biology of Blood and Marrow Transplantation
Volume 13, Issue 12 , Pages 1477-1486, December 2007

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