Optimization of Culture Conditions for the Expansion of Umbilical Cord-Derived Mesenchymal Stem or Stromal Cell-Like Cells Using Xeno-Free Culture Conditions

Abstract

First isolated from bone marrow, mesenchymal stem or stromal cells (MSC) were shown to be present in several postnatal and extraembryonic tissues as well as in a large variety of fetal tissues (e.g., fatty tissue, dental pulp, placenta, umbilical cord blood, and tissue). In this study, an optimized protocol for the expansion of MSC-like cells from whole umbilical cord tissue under xeno-free culture conditions is proposed. Different fetal calf sera and human serum (HS) were compared with regard to cell proliferation and MSC marker stability in long-term expansion experiments, and HS was shown to support optimal growth conditions. Additionally, the optimal concentration of HS during the cultivation was determined. With regard to cell proliferative potential, apoptosis, colony-forming unit fibroblast frequency, and cell senescence, our findings suggest that an efficient expansion of the cells is carried out best in media supplemented with 10% HS. Under our given xeno-free culture conditions, MSC-like cells were found to display in vitro immunoprivileged and immunomodulatory properties, which were assessed by co-culture and transwell culture experiments with carboxyfluorescein diacetate succinimidyl ester-labeled peripheral blood mononuclear cells. These findings may be of great value for the establishment of biotechnological protocols for the delivery of sufficient cell numbers of high quality for regenerative medicine purposes.

Introduction

Mesenchymal stem or stromal cells (MSC) are of great interest for cell-based therapies and tissue engineering approaches, as these cells are capable for extensive self-renewal and display a multilineage differentiation potential.^1–4 Additionally, MSC were shown to exhibit immunomodulatory properties^5–8 and display supportive function through parakrine effects; that is, MSC support tissue repair by stimulating and modulating tissue-specific cells rather than differentiating into specialized cells.^9,10 Since first isolated in the mid-1960s,¹¹ bone marrow (BM)-derived MSC (BMSC) are currently almost exclusively used in clinical trials. Nevertheless, the collecting procedure from BM is invasive and painful, and the number, differentiation potential, and maximal life span of MSC from BM decline with increasing age.^12–14 Therefore, MSC isolated from alternative and easily accessible sources gained more and more attention over the last decade. MSC were found to be present in a variety of postnatal and extraembryonic tissues and organs as well as in a large variety of fetal tissues.^15–17

The tissue of the human umbilical cord (UC) has been found to be a rich source of MSC,^18–21 and because it is easily accessible, this tissue may represent a valuable alternative to BM as a source of MSC. Pioneer works of several groups during the last decade demonstrated that the tissues of the UC harbor MSC populations exhibiting potential for clinical applications (reviewed in Ref.²²). Low level of rejection was observed in transplantation studies in animals^23–26 and first reports strongly suggest that MSC derived from UC display similar immunoprivileged properties^27–29 as described for BMSC. Given these recent findings, UC-MSC may have a great potential for autologous as well as allogeneic transplantation to initiate tissue repair.

The development of suitable biotechnological protocols for the ex vivo expansion of UC-MSC is a challenge to deliver a sufficient number of cells to a patient. MSC have been derived from UC tissue by various approaches using nonhuman serum (HS) such as fetal calf serum (FCS) for isolation and expansion of the cells. However, the use of sera of animal origin raises some safety concerns—in particular, the potential transmission of infections, for example, viruses and prions from animals to humans. In this context, the use of HS would be preferable for the delivery of cells of clinical grade.

In this work, the expansion potential as well as the stability of UC-MSC-like cells cultivated under xeno-free conditions using HS was investigated, and an optimized protocol is proposed for the cultivation of these cells. In addition, the immune properties of UC-MSC-like cells expanded under xeno-free conditions were studied to confirm the privileged immune status of the cells. Our findings may be useful for the development of a reliable biotechnological process for the delivery of MSC of clinical grade.

Materials and Methods

MSC-like cell isolation from umbilical cord tissue

Human UCs were obtained from consenting patients (n = 3) delivering full-term (38–40 weeks) infants by Cesarean section. The use of this material has been approved by the Institutional Review Board, project #3037 in an extended permission on June 17, 2006.

MSC-like cells were isolated as reported previously.³⁰ In brief, UC tissue was washed with phosphate-buffered saline (PBS), cut into ∼0.5 cm³ large pieces, and incubated in αMEM (Invitrogen GmbH) supplemented with 15% of allogeneic HS (kindly provided by the Division of Transfusion Medicine, Medical University Hannover, Germany) and 50 μg/mL gentamicin (PAA laboratories GmbH) at 37°C in a humidified atmosphere with 5% CO₂. After 2 weeks, UC tissue was removed and the adherent cells were harvested by accutase (PAA laboratories GmbH) treatment according to the manufacturer's protocol for 5 min at 37°C. The obtained cell suspension was centrifuged at 200 g for 5 min, and the cells were resuspended in αMEM supplemented with 10% HS and 50 μg/mL gentamicin and subcultured at a density of 4000 cells/cm². Following the second subconfluent passage, cells were harvested for the following experiments or cryopreserved. Cryopreservation was performed with about 1.5 × 10⁶ cells/mL in αMEM containing 10% (v/v) dimethyl sulfoxide (Sigma-Aldrich) and 80% of HS in liquid nitrogen.

Phenotypic analysis by flow cytometry

Characterization of the immunophenotype was performed as described previously.³⁰ MSC were harvested by use of accutase for 5 min at 37°C, washed twice in ice-cold PBS supplemented with 2% FCS (PAA laboratories GmbH), and resuspended to a concentration of about 10⁵ cells/antibody test. After storage for 20 min at room temperature in the dark, 400 μL of PBS supplemented with 2% FCS was added and analyzed in the EPICS XL/MCL flow cytometer (Beckman Coulter GmbH). Living cells were gated in a dot plot of forward versus side scatter signals acquired on linear scale. At least, 10,000 gated events were acquired on a LOG fluorescence scale. Positive staining was defined as the emission of a fluorescence signal that exceeded levels obtained by >99% of cells from the control population stained with matched isotype antibodies. For the antigen expression that was normalized to cell size, the fluorescence of the conjugated monoclonal antibodies as well as the forward scatter signals were measured on linear scale. The ratios of fluorescence signals versus scatter signals were calculated by the EPICS XL/MCL flow cytometer (Beckman Coulter GmbH). Histograms were generated using the software WinMDI 2.8 (Joseph Trotter).

Serum selection

The selection criteria were efficient expansion of MSC-like cells from human UC tissue and stable expression of MSC markers.

MSC-like cells were seeded at a density of 4000 cells/cm² and cultivated in 25 cm² cell culture flasks (Sarstedt) in αMEM containing 50 μg/mL gentamicin and 10% of one of the following sera: allogeneic HS, FCS standard quality, FCS gold standard, FCS pretested for amnion cells, or FCS heat-inactivated (all FCS obtained from PAA laboratories GmbH). Cells were cultivated over seven passages (P2–P8). After ∼80% of confluency in the most rapidly proliferating subculture, cells from all five cultures were simultaneously harvested and replated at the same cell density. The cell number within the individual passages was determinate by the use of phase-contrast microscopy and trypan blue exclusion test. Phenotypic analyses were performed with cells from P3, P8, and P9, respectively, by flow cytometry (see above).

Serum concentration

The selection criteria for the optimal serum concentration were cell proliferative potential, cell caspase-3/7 activity, colony-forming unit fibroblast (CFU-F) frequency, and cell senescence. The used culture media, serum concentrations, and supplements are summarized in Table 1.

Table 1.

Overview of the Used Culture Media and Supplements for Defining the Optimal Serum Concentration

Name	Formulation
HS 10	αMEM supplemented with 10% HS, 50 μg/mL gentamicin
HS 5	αMEM supplemented with 5% HS, 50 μg/mL gentamicin
HS 2	αMEM supplemented with 2% HS, 50 μg/mL gentamicin, 0.5% ITS^a, 0.5% HSA^b
HS FGF	αMEM supplemented with 2% HS, 50 μg/mL gentamicin, 0.5% ITS^a, 0.5% HSA^b, 0.5 ng/mL FGF-2

Insulin-transferrin-selenium (ITS) supplement (Invitrogen GmbH, Karlsruhe, Germany).

Human serum albumin (HSA) (DRK-Blutspendedienst, Springe, Germany).

Experiments were performed for a period of three passages (P2–P4).

For the determination of cell proliferative potential and cell caspase-3/7 activity, MSC-like cells were seeded at a density of 4000 cells/cm² in 8 wells of a 24-well cell culture plate (Sarstedt) in culture media as mentioned above. After ∼80% of confluency in the most rapidly proliferating subculture, cells from four wells of all four cultures were simultaneously harvested by accutase treatment and counted using phase-contrast microscopy and trypan blue exclusion. Cells from the remaining four wells from each culture were analyzed for caspase-3/7 activity using the Apo-ONE^® Homogeneous Caspase3-/7 Assay (Promega). Cells were washed with PBS and incubated with 200 μL of the Apo-One reagent for 1 h at room temperature in the dark. Fluorescence was recorded at 485/538 nm using a Fluoroskan Ascent Microplate Fluorometer (Thermo Fisher Scientific). The average of fluorescence values of the culture media were subtracted and values were normalized to cell numbers.

For the determination of the CFU-F frequency, MSC-like cells were seeded at a density of 50 cells/cm² in 75 cm² cell culture flasks in the corresponding culture media (see above). After 8 days of culture, cells were washed with PBS, fixed, and stained with a 0.5% solution of crystal violet in methanol for 15 min. Cells were washed with PBS and allowed to dry over night. Colonies comprising 30 or more cells were counted using phase-contrast microscopy.

The amount of senescent cells was determined by the use of the Senescence-associated β-Galactosidase (SA-β-gal) Staining Kit (Cell Signaling Technology) and 4',6-Diamidin- 2'-phenylindoldihydrochlorid (Roche Diagnostics GmbH) fluorescence counterstain in accordance to the manufacturers' instructions. For this purpose, 4000 cells/cm² were cultivated for 48 h in the corresponding media before SA-β-gal staining. After completion of the staining procedures, four representative images were taken from diverse areas of each cell culture using phase-contrast microscopy, fluorescence microscopy, and Cell^BImaging Software (Olympus GmbH). For the calculation of the percentage of senescent cells, the total number of cell nuclei and number of cell nuclei surrounded by cyan dye were enumerated.

Determination of cell in vitro immuno-modulatory properties

In vitro immuno-modulatory properties of UC-derived MSC-like cells were assessed by co-culture and transwell culture experiments with carboxyfluorescein diacetate succinimidyl ester-labeled (CFSE) peripheral blood mononuclear cells (PBMC). During all experiments, cells were cultivated in αMEM containing 50 μg/mL gentamicin and 10% HS.

PBMC were extracted from peripheral blood (kindly provided by the Division of Transfusion Medicine, Medical University Hannover, Germany). Cell separation was achieved by Lymphosep (c^.c^.pro) density gradient centrifugation.

Cells were labeled with 2 μM CFSE using the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen GmbH) at a concentration of 10⁷ cells/mL in PBS supplemented with 2% FCS for 3 min at room temperature. Staining was quenched by the addition of two volumes of culture media. PBMC were washed twice with PBS, resuspended in 2 mL culture media and counted using phase-contrast microscopy and trypan blue exclusion test.

For co-culture experiments, MSC-like cells were seeded at a density of 4 × 10⁴ cells per well in 24-well cell culture plates (Sarstedt). Once the cells had attached to the surface (∼1 h), 4 × 10⁵ CFSE-stained PBMC were transferred to the well. PBMC were harvested at days 3 and 5 of incubation to assess proliferation based on CFSE intensity by flow cytometry. Cell proliferation is marked by a decrease in fluorescence intensity due to cell division.

Transwell culture experiments were performed in 24-well cell culture plates, using Millicell culture plate inserts (Millipore). About 4 × 10⁵ CFSE-stained PBMC were placed on the bottom of the well and MSC-like cells were seeded at a density of 4 × 10⁴ cells on the membrane inserts. PBMC were harvested at days 3 and 5 of incubation to assess proliferation by flow cytometry.

For the phytohaemagglutinin (PHA) activation assay, CFSE-stained PBMC were seeded at a density of 4 × 10⁵ cells per well in 24-well cell culture plates. Cell proliferation was stimulated using PHA (2 μg/mL; Sigma) 24 h before co-culture and transwell culture assays. Experiments were performed and proliferation was assessed as described above.

Interleukin (IL)-2 concentrations were detected in cell culture supernatants of co-culture experiments using an enzyme-linked immunosorbent assay (RayBiotech, BioCat). Samples were harvested on days 1, 3, and 5.

Results

Defining optimal cell culture conditions for UC MSC-like cells: Serum selection

To investigate the expansion potential of UC MSC-like cells under xeno-free culture conditions, allogeneic HS was tested against four different FCS with regard to proliferation supportive properties. MSC-like cells were cultured over a period of seven passages starting with seeding densities of 4000 cells/cm² in αMEM supplemented with 10% of human of the respective serum. After reaching ∼80% confluency in the fastest growing culture, cells from all cultures were harvested, counted, and sub-cultured. UC-derived MSC-like cells were able to grow in the presence of all tested sera, however, with remarkable differences in cell proliferation and morphology. Cells cultivated in the presence of HS displayed the highest proliferative potential and passed 23 ± 0.31 cell population doublings in 28 days of culture, whereas all other sera lead to lower cumulative cell population doublings (Fig. 1A). Cells cultivated in FCS “heat inactivated” and FCS “pretested for amnion cells” were found to perform more cell population doublings (21 ± 0.51 and 19 ± 0.06 cell population doublings, respectively) compared to cells cultivated in the presence of FCS “standard quality” and FCS “gold standard” (13 ± 0.09 and 12 ± 0.02 cell population doublings respectively).

FIG. 1.

Influence of different sera on the proliferative activity of MSC-like cells. Proliferation was assessed by calculating cumulative cell doublings (A) and cumulative cell numbers (B). Cells were seeded at a density of 4000 cells/cm² and cultivated in αMEM containing 10% HS or one of four different FCS over seven passages (P2–P8) in duplicates. (C) Images of the cell morphologies were taken during every passage using phase-contrast microcopy. Exemplary images of cells in P8 are presented. MSC, mesenchymal stem or stromal cells; FCS, fetal calf serum; HS, human serum.

Besides their influence on the proliferative potential of the cells, the tested FCS had significant impact on morphology and size of the cells in culture. The use of FCS “gold standard” and FCS “standard quality” led to an increase in cell size (Fig. 1B) and to a more heterogenic cell population (detected by CASY^®1 DT Cell counter [innovates AG Germany]; data not shown) of cells from P8 compared to cells from P3. The use of FCS “pretested for amnion cells” and FCS “heat inactivated” had only little influence on cell morphologies. In HS cultures no remarkable morphological changes could be observed for cells in P8 as compared to P3.

MSC marker expression was found to be stable at least until P3 under any tested media formulation. Further cultivation of primary UC-MSC in FCS “standard quality” and “gold standard” until P8 was accompanied by an increase in cell size and a significant loss of MSC marker expression such as CD90, CD73, and CD105, as shown in Figure 2. In contrast, cells cultivated in HS exhibit a stable immunophenotype over the entire investigated time period. Cells cultivated with FCS “heat inactivated” and “pretested for amnion cells” were found more stable with respect to MSC marker expression (Fig. 2). Expression of the surface marker CD44 was stable under any tested media formulation (data not shown).

FIG. 2.

Stability of the immunophenotype of MSC-like cells under the influence of different sera. Cells were seeded at a density of 4000 cells/cm² and cultivated in αMEM containing 10% HS or one of four different FCS over eight passages (P2–P9) in duplicates. Marker expression was analyzed via flow cytometry on P3 and P9. Cells cultured in FCS “standard quality” and FCS “gold standard” could only be expanded until passage 8.

Optimal serum concentration

For further improvement of UC MSC-like cell growth conditions, the medium was optimized with regard to the optimal serum concentration. Additionally, the effect of the growth factor FGF-2 on the growth of the cells was analyzed. To differentiate between the effects of the serum and the growth factor on cell proliferation, apoptosis, CFU-F potential, and senescence, FGF-2-supplemented media contained only 2% HS.

MSC-like cells were cultivated for a period of three passages (P2–P4) in αMEM supplemented with HS in a concentration of 10%, 5%, 2%, and 2% + FGF-2, respectively (Table 1). The proliferative potential of the cells was determined by calculating cumulative cell population doublings. Cells were able to proliferate under all conditions. Nevertheless, remarkable differences could be observed (Fig. 3A). Cells cultivated in HS FGF displayed the highest proliferative potential and passed 8.1 ± 0.29 population doublings, whereas cells cultivated in HS 10 performed 7.9 ± 0.02, in HS 5 7.6 ± 0.01, and in HS 2 only 5.2 ± 0.21 population doublings.

FIG. 3.

Influence of the serum concentration on the growth of MSC-like cells. Cells were cultivated in αMEM supplemented with four different (HS 10, HS 5, HS 2, and HS FGF). Cells were cultivated for a period of three passages (P2–P4). Exemplary results are presented, but similar tendencies were observed in all experiments (n = 3). (A) Cumulative cell population doublings were calculated to assess the proliferative potential of MSC-like cells. Further, the amount of apoptotic cells (B), the colony-forming unit fibroblast frequency (C), and the amount of senescent cells (D) were detected.

Apoptosis in MSC-like cell cultures was determined by caspase-3/7 assays, as caspase-3 and -7 play key effector roles in apoptosis in mammalian cells. Assays were performed upon every passage (P2–P4) (Fig. 3B). Results are displayed as relative fluorescence units (RFU) (Fig. 3B). Caspase activities increased during subculture in all investigated approaches, but higher activities were detected when cells were maintained in HS 5 [1.7 ± 0.13 RFU (P2); 3.2 ± 0.35 RFU (P3); 2.7 ± 0.08 RFU (P4)] and HS 2 [1.6 ± 0.3 RFU (P2); 4.5 ± 0.4 RFU (P3); 3.7 ± 0.35 RFU (P4)]. Compared to cell cultures with HS FGF, caspase activities in the presence of HS 10 were slightly higher in P3 [2.2 ± 0.32 RFU (HS 10); 1.7 ± 0.38 RFU (HS FGF)] but remained stable on P4 (2.1 ± 0.35 RFU), whereas activities in the presence of HS FGF increased to 3.3 ± 0.39 RFU in P4.

The CFU-F frequency of MSC-like cells was determined under the same culture conditions as described for the caspase-3/7 assays. The results are described as number of colonies in 3750 seeded cells (Fig. 3C). Cells were able to form colonies under every chosen serum concentration, but most colonies were found when using HS FGF (101 P2; 163 P3; 118 P4). Cells cultivated in HS 2 displayed the lowest CFU-F frequency (81 P2; 27 P3; 14 P4), followed by cells cultured in HS 5 (83 P2; 34 P3; 25 P4) and HS 10 (84 P2; 47 P3; 38 P4), respectively.

The amount of senescent cells was determined by SA-β-gal staining and increased during cultivation under all tested conditions, but the highest amount was detected in cell cultures cultivated in HS 2 (0.41% ± 0.04% P2; 2.31% ± 0.17% P3; 24.85% ± 9.41% P4). Cells cultivated in HS 10 and HS 5 displayed similar tendencies (HS 10: 0.28% ± 0.10% P2; 0.88% ± 0.16% P3; 4.21% ± 1.41% P4) (HS 5: 0.45% ± 0.10% P2; 1.45% ± 0.60% P3; 5.02% ± 0.92% P4). Percentages of senescent cells cultivated in HS FGF were the lowest in P2 (0.15 ± 0.08) and P3 (0.41 ± 0.01) but significantly increased in P4 (13.85 ± 5.72).

In vitro immunomodulatory properties of UC MSC-like cells

In vitro immunomodulatory properties of MSC-like cells were assessed by CFSE-based proliferation assays in direct co-culture or transwell culture experiments by flow cytometry (Fig. 4A). After stimulation with PHA PBMC were analyzed on days 3 and 5. The addition of PHA led to 28.6% proliferating cells on day 3 and to 94.6% proliferating cells on day 5. Co-culture as well as transwell cultures with MSC-like cells decreased cell proliferation on day 5 with 81.9% proliferation in the transwell system and 68.8% in co-cultures with direct cell–cell contacts (Fig. 4B). The presence of MSC-like cells alone induced no proliferation of resting PBMC in either culture system (Fig. 4A “nonstimulated”).

FIG. 4.

Immunomodulatory properties of MSC-like cells. Exemplary results are presented, but similar tendencies were observed in all experiments (n = 3). (A) Flow cytometric analysis of carboxyfluorescein diacetate succinimidyl ester labeled-stained PBMC cell proliferation from direct co-culture (black area [I]) and transwell culture (dark gray area [II]) experiments. At least 10,000 events are displayed. Unless otherwise specified, PBMC proliferation was stimulated with PHA. Monocultured stimulated (gray area [III]) or nonstimulated (black lines in each histogram) PBMC served as a control. Histogram (IV) displays an overlay of histograms (I)–(III). Histogram (V) displays the results of direct co-culture (dark gray lines) and transwell culture (gray area) experiments with nonstimulated PBMC. In each histogram, marker “M3” indicates the percentage of proliferating PBMC related to the total number of nonstimulated resting PBMC (summarized in B). (C) Interleukin-2 production in direct co-culture experiments was quantitatively determined by enzyme-linked immunosorbent assay at days 1, 3, and 5. PBMC, peripheral blood mononuclear cells; PHA, phytohaemagglutinin.

Quantitative measurements of the IL-2 expression were performed using enzyme-linked immunosorbent assays. As the cytokine IL-2 is mainly produced by T-cells upon stimulation by mitogens or antigens, protein levels can be used as an additional indicator of immunomodulatory effects of UC MSC-like cells. IL-2 was quantitatively determined on days 1, 3, and 5 in supernatants of co-cultures of stimulated and nonstimulated PBMC, monocultured stimulated and nonstimulated PBMC, and monocultured MSC-like cells. Cells of the latter two approaches displayed only low expression of IL-2 (20 pg/mL), whereas PHA-stimulated PBMC produced significantly more IL-2 (387 ± 3.9 pg/mL on day 1, 669 ±5.5 pg/mL on day 3, and 628 ± 11.2 pg/mL on day 5). In contrast, considerably lower IL-2 amounts were detected in co-cultures of stimulated PBMC and MSC-like cells (267 ± 3.6 pg/mL on day 1, 116 ± 5.4 pg/mL on day 3, and 58 ± 0.8 pg/mL on day 5). No significant differences in IL-2 secretion could be observed between monocultured nonstimulated PBMC and co-cultured nonstimulated PBMC.

Discussion

UC-MSC bear high potential for applications in regenerative medicine. Nevertheless, delivering sufficient numbers of high quality cells to patients is one of the major challenges. Thus, suitable biotechnological protocols need to be developed for the ex vivo expansion of UC-MSC. To our knowledge, we are the first to describe the expansion of MSC-like cells from whole human UC tissue under xeno-free culture conditions. We here demonstrate that the culture medium containing HS was associated with an elevated proliferative potential of the cells. In contrast to the use of HS, at least two of the tested FCS led to inhomogeneous cell populations displaying strong morphological and phenotypic changes with a loss of typical stem cell markers. Together, these data suggested several advantages for a stable UC-MSC maintenance by the use of HS in xeno-free culture conditions. Indeed, recent studies substantiated a xeno-free environment demonstrating that UC-MSC can be stably expanded and in addition, can be successfully differentiated in HS-supplemented media.^31,32

To further optimize the culture conditions, optimal serum concentrations and the proliferation-supporting effects of FGF-2 were determined. Based on the cell proliferative potential, the cell caspase-3/7 activity, the CFU-F frequency, and the amount of senescent cells, our findings suggest that the expansion of MSC-like cells is carried out best in αMEM supplemented with 10% HS. Although cells displayed the highest proliferative capacity and CFU-F frequency in the presence of FGF-2, a drastic increase in caspase-3/7 activity and percentage of senescent cells could be observed during cultivation (P4). Caspase activity further increased upon switching from FGF-2 supplemented media to media containing only 10% HS in P5 (data not shown). These findings indicate that media for a long-term ex vivo expansion of MSC-like cells of high quality should devoid FGF-2. Under any tested media formulation, an increase of caspase-3/7 activity could be observed from P2–P3 followed by a decrease from P3–P4. Due to our stringent culture conditions, a selection of distinct subpopulations within the primary culture might have occurred. However, further studies are required to evaluate this hypothesis.

Under our given xeno-free culture conditions, MSC-like cells were found to display in vitro immunoprivileged properties, as no proliferation of resting PBMC was induced in direct co-culture or indirect transwell culture experiments. Further, MSC-like cells exhibit in vitro immunomodulatory properties, as PHA-stimulated proliferation of PBMC was decreased in both, transwell and direct co-culture experiments. This indicates, that immunomodulation is mediated by direct cell-to-cell contacts as well as by soluble factors. Besides T cells, several other immuno-competent cell types were found to be influenced by MSC (e.g., B lymphocytes, natural killer cells, and dendritic cells) and several factors and enzymes have been proposed to be involved in immunomodulation, including prostaglandin E2, indoleamine 2,3-dioxygenase, human leukocyte antigen G (HLA-G), insulin-like growth factor-binding proteins, transforming growth factor-β, hepatocyte growth factor, and nitric oxide.^8,33,34 Our findings indicate that the cell-to-cell contact plays a central role in immunomodulation, as a stronger inhibitory effect on PBMC proliferation could be observed in direct co-culture experiments. Similar results were described by other groups examining the immunomodulatory properties of BMSC.^35–37

In this work, the efficient ex vivo expansion of UC-derived MSC-like cells under xeno-free conditions was demonstrated. Our results reveal that a long-term expansion of the cells is optimal in the presence of HS supporting high proliferative potential, a homogenous morphology, a stable MSC marker expression, and sustained immunomodulatory properties. These findings may be of high interest for the establishment of biotechnological protocols for generating high-quality cell material for regenerative medicine purposes.

Footnotes

Acknowledgments

The authors would like to thank Dr. Magda Tomala for technical assistance, Prof. Dr. Britta Eiz-Vesper and Prof. Dr. Rainer Blasczyk (Division of Transfusion Medicine, Medical University Hannover, Germany) for providing PBMC, and all three for fruitful discussion.

This work was supported by the German Research Foundation through project funding KA 1784/5 and funding of Cluster of Excellence “REBIRTH”.

Disclosure Statement

No competing financial interests exist.

References

Choong

P.F.

, Mok

P.L.

, Cheong

S.K.

, Leong

C.F.

, Then

K.Y.

Generating neuron-like cells from BM-derived mesenchymal stromal cells in vitro. Cytotherapy, 9:170. 2007.

Pittenger

M.F.

, Mackay

A.M.

, Beck

S.C.

, Jaiswal

R.K.

, Douglas

, Mosca

J.D.

et al. Multilineage potential of adult human mesenchymal stem cells. Science, 284:143. 1999.

Saulnier

, Lattanzi

, Puglisi

M.A.

, Pani

, Barba

, Piscaglia

A.C.

et al. Mesenchymal stromal cells multipotency and plasticity: induction toward the hepatic lineage. Eur Rev Med Pharmacol Sci, 13,Suppl 1:71. 2009.

, Jiang

, Guo

, Chen

, Zou

, Ke

et al. Functional analysis of neuron-like cells differentiated from neural stem cells derived from bone marrow stroma cells in vitro. Cell Mol Neurobiol, 28:545. 2008.

Kode

J.A.

, Mukherjee

, Joglekar

M.V.

, Hardikar

A.A.

Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration. Cytotherapy, 11:377. 2009.

Le Blanc

Immunomodulatory effects of fetal and adult mesenchymal stem cells. Cytotherapy, 5:485. 2003.

Le Blanc

, Ringden

Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation. Biol Blood Marrow Transplant, 11:321. 2005.

Siegel

, Schafer

, Dazzi

The immunosuppressive properties of mesenchymal stem cells. Transplantation, 87:S45. 2009.

Caplan

A.I.

Why are MSCs therapeutic? New data: new insight. J Pathol, 217:318. 2009.

10.

Meirelles Lda

, Fontes

A.M.

, Covas

D.T.

, Caplan

A.I.

Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev, 20:419. 2009.

11.

Friedenstein

A.J.

Osteogenetic activity of transplanted transitional epithelium. Acta Anat (Basel), 45:31. 1961.

12.

Mueller

S.M.

, Glowacki

Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges. J Cell Biochem, 82:583. 2001.

13.

Nishida

, Endo

, Yamagiwa

, Tanizawa

, Takahashi

H.E.

Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation. J Bone Miner Metab, 17:171. 1999.

14.

Stenderup

, Justesen

, Clausen

, Kassem

Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone, 33:919. 2003.

15.

da Silva Meirelles

, Chagastelles

P.C.

, Nardi

N.B.

Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci, 119:2204. 2006.

16.

Marcus

A.J.

, Woodbury

Fetal stem cells from extra-embryonic tissues: do not discard. J Cell Mol Med, 12:730. 2008.

17.

Pappa

K.I.

, Anagnou

N.P.

Novel sources of fetal stem cells: where do they fit on the developmental continuum?

Regen Med, 4:423. 2009.

18.

Erices

, Conget

, Minguell

J.J.

Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol, 109:235. 2000.

19.

Romanov

Y.A.

, Svintsitskaya

V.A.

, Smirnov

V.N.

Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells, 21:105. 2003.

20.

Mitchell

K.E.

, Weiss

M.L.

, Mitchell

B.M.

, Martin

, Davis

, Morales

et al. Matrix cells from Wharton's jelly form neurons and glia. Stem Cells, 21:50. 2003.

21.

Sarugaser

, Lickorish

, Baksh

, Hosseini

M.M.

, Davies

J.E.

Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells, 23:220. 2005.

22.

Moretti

, Hatlapatka

, Marten

, Lavrentieva

, Majore

, Hass

et al. Mesenchymal stromal cells derived from human umbilical cord tissues: primitive cells with potential for clinical and tissue engineering applications. Adv Biochem Eng Biotechnol, 123:29. 2010.

23.

Yang

C.C.

, Shih

Y.H.

, Ko

M.H.

, Hsu

S.Y.

, Cheng

, Fu

Y.S.

Transplantation of human umbilical mesenchymal stem cells from Wharton's jelly after complete transection of the rat spinal cord. PLoS ONE, 3:e3336. 2008.

24.

Weiss

M.L.

, Medicetty

, Bledsoe

A.R.

, Rachakatla

R.S.

, Choi

, Merchav

et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells, 24:781. 2006.

25.

Liao

, Xie

, Zhong

, Liu

, Du

, Zhou

et al. Therapeutic effect of human umbilical cord multipotent mesenchymal stromal cells in a rat model of stroke. Transplantation, 87:350. 2009.

26.

Koh

S.H.

, Kim

K.S.

, Choi

M.R.

, Jung

K.H.

, Park

K.S.

, Chai

Y.G.

et al. Implantation of human umbilical cord-derived mesenchymal stem cells as a neuroprotective therapy for ischemic stroke in rats. Brain Res, 1229:233. 2008.

27.

Ennis

, Gotherstrom

, Le Blanc

, Davies

J.E.

In vitro immunologic properties of human umbilical cord perivascular cells. Cytotherapy, 10:174. 2008.

28.

Weiss

M.L.

, Anderson

, Medicetty

, Seshareddy

K.B.

, Weiss

R.J.

, VanderWerff

et al. Immune properties of human umbilical cord Wharton's jelly-derived cells. Stem Cells, 26:2865. 2008.

29.

Yoo

K.H.

, Jang

I.K.

, Lee

M.W.

, Kim

H.E.

, Yang

M.S.

, Eom

et al. Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol, 259:150. 2009.

30.

Majore

, Moretti

, Hass

, Kasper

Identification of subpopulations in mesenchymal stem cell-like cultures from human umbilical cord. Cell Commun Signal, 7:6. 2009.

31.

Lavrentieva

, Majore

, Kasper

, Hass

Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells. Cell Commun Signal, 8:18. 2010.

32.

Majore

, Moretti

, Stahl

, Hass

, Kasper

Growth and differentiation properties of mesenchymal stromal cell populations derived from whole human umbilical cord. Stem Cell Rev, 2010, 10.1007/s12015.010-9165-y

33.

Hoogduijn

M.J.

, Popp

, Verbeek

, Masoodi

, Nicolaou

, Baan

et al. The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy. Int Immunopharmacol, 10:1496. 2010.

34.

Yagi

, Soto-Gutierrez

, Parekkadan

, Kitagawa

, Tompkins

R.G.

, Kobayashi

et al. Mesenchymal stem cells: mechanisms of immunomodulation and homing. Cell Transplant, 19:667. 2010.

35.

Jiang

X.X.

, Zhang

, Liu

, Zhang

S.X.

, Wu

, Yu

X.D.

et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood, 105:4120. 2005.

36.

Sotiropoulou

P.A.

, Perez

S.A.

, Gritzapis

A.D.

, Baxevanis

C.N.

, Papamichail

Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells, 24:74. 2006.

37.

Tse

W.T.

, Pendleton

J.D.

, Beyer

W.M.

, Egalka

M.C.

, Guinan

E.C.

Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation, 75:389. 2003.