Immune Properties of Human Umbilical Cord ... - Wiley Online Library

TISSUE-SPECIFIC STEM CELLS Immune Properties of Human Umbilical Cord Wharton’s Jelly-Derived Cells MARK L. WEISS,a CAMERON ANDERSON,a SATISH MEDICETTY,c KIRAN B. SESHAREDDY,a RITA J. WEISS,a IRENE VANDERWERFF,a DERYL TROYER,a KEVIN R. MCINTOSHb a

Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas, USA; bCognate BioServices, Baltimore, Maryland, USA; cNeoStem, Inc., Boston, Massachusetts, USA Key Words. Adherent cells • Immunogenicity • Immunosuppression • In vitro culture • Stromal cell

ABSTRACT Cells isolated from Wharton’s jelly, referred to as umbilical cord matrix stromal (UCMS) cells, adhere to a tissue-culture plastic substrate, express mesenchymal stromal cell (MSC) surface markers, self-renew, and are multipotent (differentiate into bone, fat, cartilage, etc.) in vitro. These properties support the notion that UCMS cells are a member of the MSC family. Here, the immune properties of UCMS cells are characterized in vitro. The overall hypothesis is that UCMS cells possess immune properties that would be permissive to allogeneic transplantation. For example, UCMS cells will suppress of the proliferation of “stimulated” lymphocytes (immune suppression) and have reduced immunogenicity (e.g., would be poor stimulators of allogeneic lymphocyte proliferation). Hypothesis testing was as follows: first, the effect on proliferation of coculture of mitotically inactivated human UCMS cells with concanavalin-A-stimulated rat splenocytes was assessed in three different assays. Second, the effect of human UCMS cells on one-way and two-way mixed lymphocyte reaction (MLR) assays was determined. Third, the expression of human leukocyte antigen (HLA)-G was examined in human UCMS cells using reverse transcription-polymerase chain reaction, since HLA-G expression conveys immune regulatory properties at the ma-

ternal-fetal interface. Fourth, the expression of CD40, CD80, and CD86 was determined by flow cytometry. Fifth, the cytokine expression of UCMS cells was evaluated by focused gene array. The results indicate that human UCMS cells inhibit splenocyte proliferation response to concanavalin A stimulation, that they do not stimulate T-cell proliferation in a one-way MLR, and that they inhibit the proliferation of stimulated T cells in a two-way MLR. Human UCMS cells do not inhibit nonstimulated splenocyte proliferation, suggesting specificity of the response. UCMS cells express mRNA for pan-HLA-G. UCMS cells do not express the costimulatory surface antigens CD40, CD80, and CD86. UCMS cells express vascular endothelial growth factor and interleukin-6, molecules previously implicated in the immune modulation observed in MSCs. In addition, the array data indicate that UCMS cells make a cytokine and other factors that may support hematopoiesis. Together, these results support previous observations made following xenotransplantation; for example, there was no evidence of frank immune rejection of undifferentiated UCMS cells. The results suggest that human UCMS will be tolerated in allogeneic transplantation. STEM CELLS 2008;26:2865–2874

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cell Therapy has suggested three defining criteria for mesenchymal stromal cells (MSCs). MSCs (a) are a plastic-adherent cell population, (b) have specific surface markers (e.g., CD14-, CD34-, CD45-, and human leukocyte antigen [HLA] class II-negative and CD73-, CD29-, CD105-positive), and (c) have the capacity to self-renew and differentiate into various lineages, including bone, cartilage, and adipose, in vitro [1]. MSCs possess the immune properties of immune suppression and immune avoidance (reviewed in [2, 3]). MSCs exert immunosuppression by inhibiting T-cell responses to polyclonal

stimuli [4 – 6]. MSCs suppress lymphocyte proliferation in vitro and prolong skin graft survival [4]. Two different general mechanisms, one cell contact-dependent and one -independent, have been advanced to explain this immunosuppression capability [7]. The effect is most likely due to soluble factors secreted by the MSCs [8], such as transforming growth factor ␤-1, hepatocyte growth factor, interleukin-6, prostaglandin E2, indoleamine 2,3-dioxygenase-mediated tryptophan depletion, or nitric oxide (reviewed in [9]). MSCs downregulate the interleukin (IL)-2 receptor (CD25) and CD38 on phytohemagglutinin-activated lymphocytes [10]. Dendritic cells (DCs) cocultured with MSCs develop into tolerogenic DCs [11]. In addition to MSCs derived from bone marrow, MSCs from other tissues are reported to be immunosuppressive (e.g., MSCs

Author contributions: M.L.W.: conception and design, assembly of data, data analysis and interpretation, manuscript writing and final approval of manuscript, financial and administrative support; C.A. and K.R.M.: design, assembly of data, data analysis and interpretation, manuscript writing; S.M., K.B.S., R.J.W., and I.V.: collection and assembly of data; D.T.: administrative support, data interpretation. Correspondence: Mark L. Weiss, Ph.D., Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506, USA. Telephone: 785-532-4520; Fax: 785-532-4557; e-mail: [email protected] Received December 10, 2007; accepted for publication July 28, 2008; first published online in STEM CELLS EXPRESS August 14, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/ stemcells.2007-1028

STEM CELLS 2008;26:2865–2874 www.StemCells.com

Immune Properties of UCMS Cells

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from dental pulp [12], adipose tissue [13, 14], and amniotic tissue [15]). The immune-suppressive property of MSCs has been exploited for the successful treatment of severe human graft-versus-host disease [16]. MSCs have immune avoidance mechanisms that reduce immunogenicity. Reduced immunogenicity is suggested by the lack of acute rejection response by the host following xenogeneic transplantation into immune-competent animals [17, 18]. Mechanisms that may allow MSCs to escape from the immune system in allogeneic hosts include modulation of host dendritic and T-cell function and promotion of regulatory T-cell induction, as well as limited expression of alloantigen [19]. Low immunogenicity is also suggested to be due to the induction of divisional anergy in T cells [6], as well as secretion of the soluble factors mentioned above by MSCs [19]. Coculture of bone marrow MSCs with T lymphocytes downregulated T-cell alloresponsiveness via the TH2 path and resulted in the T cells assuming a T-regulatory phenotype [20]. Adipose-derived MSCs demonstrate reduced immunogenicity [13, 14]. Thus, the immune properties described above are characteristics of MSCs derived from several tissue sources. We identified a population of CD45-, CD34-, and HLA-DRnegative and CD73-, CD105-, CD90-, and CD29-positive cells derived from human umbilical cord Wharton’s jelly, termed umbilical cord matrix stromal (UCMS) cells [21–24]. Human UCMS cells can be isolated rapidly in large numbers from ⬎90% of human cords. Thus, UCMS cells may be a robust source of MSC-like cells for therapeutic use since they can be frozen/thawed, clonally expanded, engineered to express exogenous proteins, and extensively expanded in culture. We speculate that UCMS cells have therapeutic potential since they resemble MSCs in many respects, including surface phenotype, plastic adherence, and multipotency; in the latter case, they can be induced in vitro to become bone, cartilage, adipose cells [22, 25–30], neural cells [22, 30, 31], skeletal muscle cells [25], and, possibly, types of muscle cells [30, 32]. Following xenotransplantation of pig or human UCMS cells into immune-competent rats, little to no host immune cell infiltration was observed, suggesting low immunogenicity of UCMS cells [21, 23, 24]. Our hypothesis is that UCMS cells have immune properties that are permissive to allogeneic transplantation. MSCs derived from bone marrow [2, 4, 5, 16, 33, 34] and adipose tissue [13, 14] are immune-suppressive and have low immunogenicity in vitro. Our hypothesis would be supported if UCMS cells share these properties. Furthermore, in some tissues, such as the chorionic plate of the placenta and perhaps parts of the umbilical cord, additional immune avoidance mechanisms are found, because the early embryo must avoid immune-based destruction, especially in hemochorial species. One well-characterized mechanism is the expression by the trophoblasts of a nonclassic HLA class I gene, HLA-G. Six or seven splice variants of HLA-G have been identified: HLA-G1 through HLA-G4 are membrane-bound isoforms; HLA-G5 and HLA-G6 are soluble forms. The soluble forms of HLA-G have been shown to have immunoregulatory functions, including inhibition of T-cell activation [35]. Although HLA-G expression is downregulated and not found in the fetus, its expression in the umbilical cord is unknown. The hypothesis would be supported if UMCS cells express HLA-G. Here, the immune properties of human UCMS cells were assessed in vitro using standard assays, including splenocyte proliferation assays and one-way and two-way mixed lymphocyte assays, and their expression of HLA-G isoforms were assessed using reverse transcription (RT)-polymerase chain reaction (PCR). The expression of costimulatory molecules CD40, CD80, and CD86 by UCMS cells was characterized by flow cytometry, and their expression of cytokines was examined by

focused gene array. The results indicate that UCMS cells are immunosuppressive, have reduced immunogenicity, express HLA-G, do not express costimulatory molecules, and express cytokines that may modulate immune function. Together, these results suggest that human UCMS will be tolerated in allogeneic transplantation.

MATERIALS

AND

METHODS

Approvals The Kansas State University Institutional Review Board reviewed and approved this protocol. The Kansas State University Institutional Animal Care and Use Committee reviewed and approved animal use.

Human Umbilical Cord Matrix Stromal Cells The protocol for isolation and characterization of umbilical cord matrix stromal cells has been previously described [22, 24]. Using previously described protocols, a subset of the isolates was characterized to confirm that they met the ISCT minimal marker set for MSCs (e.g., they were shown to be plastic-adherent and to express surface markers CD105, CD90, and CD44) and that they did not express surface markers CD34 and CD45, and they were verified to differentiate along two or three mesenchymal lineages (e.g., chondrogenic, osteogenic, and adipogenic lineages) [22, 28, 29, 36].

Splenocyte Proliferation Assay Rat splenocytes were obtained from euthanatized rats using standard protocols. Briefly, the spleen was removed aseptically, washed thoroughly, and diced, and spleen cells were released by trituration. The splenocyte preparation was cleaned up by centrifugation and resuspending the pellet in RPMI 1640 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and the erythrocytes were lysed. The splenocyte preparation was again centrifuged, and splenocytes were washed two more times with RPMI. Cell count and viability were assessed by trypan blue exclusion prior to plating. Cells were resuspended in splenocyte medium (i.e., RPMI containing 10% fetal bovine serum [FBS; HyClone, Logan, UT, http://www.hyclone. com], 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM pyruvate, 20 mM HEPES, and 5 ⫻ 10⫺5 M 2-mercaptoethanol). Human umbilical cord matrix stromal (hUCMS) cells were plated in a 12-well plate in low-serum medium (as described previously). At 70% confluence, the medium was removed, and fresh medium containing mitomycin-C (20 ␮g/ml) was added for 2 hours at 37°C to mitotically inactivate the hUCMS cells, followed by two washes and the addition of low-serum medium. In some cases, the hUCMS cells were inactivated by irradiation (7–9 Gy; Kansas State University CVM linear accelerator). Several hours later or the next day, the medium was removed from the inactivated hUCMS cells, and 1 ml of splenocyte medium containing 5 ⫻ 104 concanavalin A (ConA)-treated (10 ␮g/ml) splenocytes was added to three wells with hUCMS cells and three wells without hUCMS cells (each trial was done in triplicate, and each trial was performed with a different splenocyte isolate).

Assessing Splenocyte Proliferation Splenocyte proliferation was assessed three different ways. First, live splenocytes were counted using a hemocytometer 3 days after plating using trypan blue exclusion. Live cell splenocyte counts were done in triplicate and averaged for each well. Here, the effect of hUCMS cell coculture on splenocyte proliferation was evaluated by comparing the number of viable splenocytes in wells cocultured with inactivated hUCMS cells with the number of viable splenocytes in control wells (wells without an hUCMS cell layer; data were averaged from five independent trials). Second, in completely independent experiments, a colorimetric assay using tetrazole reduction was used to assess splenocyte proliferation following Con-A stimulation using the manufacturer’s protocols (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium [MTT]

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Figure 1. Immune suppression by coculture of human umbilical cord matrix stromal (hUCMS) cells with ConA-activated splenocytes. (A): Using the trypan blue method to determine the number of live cells, the average suppression in splenocyte growth when cocultured with hUCMS cells was 40%. (B): Using the MTT assay, the average suppression after 4 days was 25%. Thus, both methods indicated immune suppression by coculture with mitotically inactivated hUCMS cells. Data are presented as means ⫾ SE. Statistically significant differences are indicated. Abbreviations: ConA, concanavalin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; UCMS, umbilical cord matrix stromal.

assay; Roche Diagnostics, Basel, Switzerland, http://www.rocheapplied-science.com). The plate was evaluated on an enzyme-linked immunosorbent assay reader at 550 – 690 nm. The effect of hUCMS cells on splenocyte proliferation was evaluated by comparing the optical density (OD) in wells cocultured on inactivated hUCMS cells with the OD of splenocytes grown without an inactivated hUCMS cell layer (performed in triplicate and averaged from four independent trials). Third, in independent trials, the effect of coculture with inactivated hUCMS cells on Con-A-stimulated splenocytes was assessed followed dye-loading of splenocytes with carboxyfluorescein succinimidyl ester (CFSE). Briefly, splenocytes were incubated with 5 ␮M CFSE for 10 minutes followed by two washes with phosphatebuffered saline (PBS). The CFSE-labeled splenocytes were equally divided into four experimental groups: splenocytes alone (no Con-A stimulation), splenocytes cultured with mitotically inactivated hUCMS cells in splenocyte medium (no Con-A), splenocytes stimulated with Con-A (10 ␮g/ml; no hUCMS cells), and splenocytes stimulated with Con-A cultured with inactivated hUCMS cells. Twenty-four or 48 hours later the splenocytes were lifted, washed with PBS, fixed with 4% paraformaldehyde, rinsed twice with PBS, and stored at 4°C until flow cytometry (FACSCalibur; BD Biosciences, San Diego, http://www.bdbiosciences.com). The flow cytometry data were analyzed using ModFit (Verity Software House, Topsham, ME, http://www.vsh.com) to estimate the proliferation of the splenocytes. To compare the effects of the experimental variables, the proliferation index, a statistic generated by ModFit that relates to the number of population doublings the splenocytes had undergone following CFSE loading, was used. In each case, a paired t test was used to compare means of experimental groups to evaluate whether Con-A exposure increased splenocyte proliferation (CFSE experiment; one-tailed test) and whether culture with UCMS cells inhibited Con-A induced splenocyte proliferation (all three experiments; one-tailed test). A twotailed test was used to evaluate whether culture of UCMS cells with splenocytes affected splenocyte proliferation (compared with splenocytes only; CFSE experiment).

Mixed Lymphocyte Proliferation Assays The mixed lymphyocyte proliferation assays were carried out using standard methods. The methodological details are found in the supplemental online data.

RT-PCR RT-PCR was carried out using standard methods. The methodological details are found in the supplemental online data.

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Flow Cytometry Three different hUCMS cell isolates (isolates 19, 12, and 21) were assessed via flow cytometry using standard methods described previously [24]. Briefly, approximately 1–2 ⫻ 106 cells were suspended in 2 ml of PBS containing 0.5% bovine serum albumin and 2 mM EDTA. Aliquots of cells (100 ␮l) were incubated with labeled antibody (or isotype control) for 30 minutes at 4°C then washed twice by centrifugation. The cells were protected from light and held at 4°C until analysis using a FACSCalibur. The antibodies used here can be found in supplemental online Table 3.

Focused Array Nonradioactive GeArray cDNA expression array filters (OHS-022; SuperArray Bioscience Corporation, Frederick, MD, http://www. superarray.com) were used to evaluate cytokine gene expression, and procedures were as described by the manufacturer. The methodological details are provided in the supplemental online data.

Statistical Analysis Data collection was conducted in an experimenter-blind fashion, when possible. Following data collection, the group status was decoded prior to statistical analysis. In general, the Student’s t test was used to evaluate the differences in the means between groups, and significance was set at p ⬍ .05. For the CFSE experiment, significance was set at p ⬍ .1. Data are presented as mean ⫾ 1 SEM or mean ⫾ 1 SD, as noted.

RESULTS Characterization of hUCMS Cells Human UCMS cells were rapidly extracted from the umbilical cord and grown in culture. As shown in supplemental online Table 1, the umbilical cord isolates yielded an average of approximately 10,500 cells per centimeter of umbilical cord length in the initial isolate; this fits within the 10,000 –15,000 cells per centimeter of length that has historically been obtained in our laboratory [37, 38]. Here, a randomly selected subset of the isolates was fully characterized by plastic adherence, flow cytometry, and differentiation into mesenchymal lineages: osteogenic, chondrogenic, and adipogenic lineages (supplemental online Table 1). Thus, hUCMS cells used here the minimal definition of MSCs given by ISCT [1], as previously described by our laboratory and others [22, 28, 29, 36].

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Figure 2. Immune suppression by coculture of human umbilical cord matrix stromal cells (hUCMS cells) with ConA-activated splenocytes assessed by the CFSE method. Splenocyte proliferation was assessed after 4 days of coculture with hUCMS cells and analyzed by flow cytometry. The proliferation index (PI; a statistic generated by ModFit) correlated to the number of cell divisions the splenocytes had undergone. (A): Data from one of the three independent trials. Top left: Splenocytes stimulated with ConA proliferated extensively (PI, 3.63; no coculture). Top right: Coculture of stimulated splenocytes with inactivated hUCMS cells decreased splenocyte proliferation (PI, 2.63). Bottom left: Coculture of unstimulated splenocytes with hUCMS cells tended to increase, and did not decrease, unstimulated splenocyte proliferation (compare PI of this panel, 1.57, with the PI of splenocytes alone [bottom right], 1.46). Bottom right: Unstimulated splenocytes alone (PI, 1.46). (B): The data were normalized to the PI of splenocytes only (100%). Shown are averaged results from three independent trials. Splenocyte proliferation was significantly enhanced by ConA (p ⬍ .001). In contrast, culture of inactivated hUCMS cells with ConA-stimulated splenocytes significantly suppressed splenocyte proliferation (p ⬍ .08). The immune suppression effect of inactivated hUCMS cells was specific to stimulated splenocytes, and coculture with inactivated hUCMS cells was not in itself harmful to splenocytes because coculture of hUCMS cells did not affect proliferation of splenocytes (p ⬎ .1). Data are presented as means ⫾ SE. Statistically significant differences are indicated with an asterisk (ⴱ). Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; ConA, concanavalin A; NS, not significant; UCMS, umbilical cord matrix stromal.

Splenocyte Proliferation Assay Results Both the trypan blue (shown in Fig. 1A) and MTT assay (Fig. 1B) methods indicated significant suppression of Con-A-induced splenocyte proliferation by coculture with mitotically inactivated human UCMS cells. The average suppression in splenocyte growth when cocultured with UCMS cells was 40% (Fig. 1A). Using the MTT assay, immune suppression ranged from 8% to 69% after 3 days and from 15% to 55% after 4 days. The average suppression was 30% after 3 days and 25% after 4 days (Fig. 1B). Thus, both methods indicated significant suppression of splenocyte proliferation by coculture with inactivated hUCMS cells. The MTT assay results indicated that suppression was 25% after 4 days versus 40% suppression of proliferation obtained by manual counting after 4 days. The CFSE experiment was conducted to evaluate splenocyte proliferation and to evaluate the specificity of the effect of hUCMS on activated splenocytes. The results are shown in Figure 2 and are similar to those obtained using the other methods; for example, they indicate a suppression of Con-Ainduced splenocyte proliferation by coculture with mitotically inactivated hUCMS cells (Fig. 1). Specifically, splenocytes grown in culture without the mitogen Con-A yielded a proliferation index of 2.17 ⫾ 0.77. Stimulation with Con-A significantly increased the proliferation of splenocytes (proliferation index, 5.38 ⫾ 2.17). This indicates that the splenocytes proliferate in response to Con-A stimulation (positive control, p ⬍

.01). The coculture of Con-A-stimulated splenocytes with mitotically inactivated hUCMS cells produced a proliferation index of 3.80 ⫾ 1.08. This represents a significant suppression of activated splenocyte proliferation (p ⬍ .08). To evaluate whether coculture of splenocytes with inactivated UCMS cells produced a nonspecific inhibition of splenocyte proliferation, the proliferation index was assessed following coculture of unstimulated (no Con-A stimulation) splenocytes with inactivated hUCMS cells. In this case, the proliferation index was 2.39 ⫾ 1.38. Thus, splenocyte proliferation is mildly stimulated (and not significantly stimulated) and was not inhibited by coculture with human UCMS cells. This observation suggests that the suppressive effect of hUCMS cells on splenocytes is specific to mitogen-induced splenocyte proliferation. These results provide independent confirmation of the data obtained from by manual counting and by MTT assays and extend those findings to indicate that (a) hUCMS cells lack toxicity on splenocytes when cocultured and (b) the immune-suppressive effects of UCMS cells are specific to splenocytes stimulated by Con-A.

One-Way Mixed Lymphocyte Reaction (Immunogenicity Assay) Results The data shown in Figure 3A and 3B are derived from independent experiments performed on different cell isolates. In both cases, the experiments were performed the same way. As indi-


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Figure 3. Immunogenicity of human umbilical cord matrix stromal (hUCMS) cells by one-way mixed lymphocyte reaction assay. Four different hUCMS cell isolates at early (P5–P6) or later (P9) passage were tested for their ability to stimulate proliferation of allogeneic T cells derived from two different donors, responder 008 and responder 009. The proliferation of T cells was determined in the presence of autologous irradiated PBMCs (negative control, background), in the presence of allogeneic irradiated PBMCs (positive control), or in the presence of UCMS cells. Results are shown for hUCMS donors 4-3 and 4-5 in (A) and for hUCMS donors 4-6 and 4-7 in (B). The stimulator cells were tested at densities of 5,000, 10,000, or 20,000 cells per well. Only the highest cell dose is shown here since the lower doses of positive control allogeneic PBMCs induced poor T-cell proliferation. Data are presented as mean counts per minute ⫾ SD. An asterisk (ⴱ) indicates significant T-cell stimulation above background responses by criteria described in Materials and Methods. Abbreviations: P, passage; PBMC, peripheral blood mononuclear cell; UCMS, umbilical cord matrix stromal.

cated in Figure 3A and 3B, the purified T-cell response to hUCMS cell isolates derived from four donors was not significantly higher than the response to autologous peripheral blood mononuclear cells (PBMCs), indicating that hUCMS cells did not elicit a proliferation response in vitro. Importantly, the response of all four isolates did not change with passage: neither early passage (P5 or P6) nor later passage (P9) cells elicited an appreciable T-cell proliferation response. In contrast, vigorous T-cell responses were detected to allogeneic PBMCs (the positive control).

Two-Way Mixed Lymphocyte Reaction (Immune Suppression) Results The data shown in Figure 4A and 4B were derived from independent experiments performed on different cell isolates. In both cases, the experiments were performed the same way. As shown in Figure 4A and 4B, all four hUCMS cell isolates suppressed the mixed lymphocyte reaction (MLR) response to various degrees, generally in a dose-dependent manner. In the first set of experiments (Fig. 4A), control splenic fibroblasts were not suppressive at the highest dose of 20,000 cells per well. At the same dose, hUCMS cells suppressed the MLR by an average of 35% ⫾ 14% (range, 18%–53%). This dose of cells corresponds to a 1:10 ratio of hUCMS to responder cells, assuming that half of the PBMCs are T cells. In the second set of experiments (Fig. 4B), splenic fibroblasts were suppressive at 10,000 cells per well (34% suppression) but not at 5,000 cells per well (8% suppression). At 5,000 cells per well, hUCMS cells suppressed the MLR by an average of 36% ⫾ 15% (range, www.StemCells.com

19%– 49%). This dose of cells corresponds to a 1:40 ratio of hUCMS to responder cells. The immune suppression data from both sets of experiments is summarized in Figure 5, comparing passage number of UCMS cells. In two of three isolates (4-3, 4-5) suppression decreased with increased passage by approximately 19%. Immune suppression by the third hUCMS cell isolate (4-7) was unchanged by passage. Overall suppression by early passage (P5–P6) hUCMS cells was 37% ⫾ 14%, and suppression by late passage (P9) cells was 33% ⫾ 15%. The immune suppression observed by allogeneic MLR (approximately 35%) was similar to that observed by the xenogeneic splenocyte proliferation assays (Figs. 1 and 2, above).

RT-PCR Results Pan-HLA-G primers indicated that HLA-G mRNA was expressed by hUCMS cells at both passage 4 and passage 8 (data not shown). As shown in Figure 6, hUCMS cells differed from the term placenta in terms of the expression of mRNA for HLA-G6 isoforms. Human UCMS cells weakly expressed HLA-G6 and did not express HLA-G5 (Fig. 6). In contrast, term placenta expressed both HLA-G5 and HLA-G6. Data from the same PCR experiment depicting results from the JAR p745 negative control cells, and the jeg-3 HLA-G positive controls are shown as well.

Flow Cytometry Results As shown in Table 1, three different isolates of hUCMS cells tested at passage 4 did not express costimulatory molecules (e.g., CD40, CD80, or CD86).

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Figure 4. Suppression of two-way MLR by human umbilical cord matrix stromal (hUCMS) cells. Four different hUCMS cell isolates were tested at early (P5 or P6) or later (P9) passage for their ability to suppress T-cell proliferation in a two-way MLR. hUCMS cells or splenic fibroblasts were added to MLR cultures at the numbers indicated, ranging from 5,000 to 20,000 cells per well. T-cell proliferation, expressed as cpm, was determined 7 days later by pulsing the cells with [3H]thymidine during the final 16 hours of culture. Data are represented as mean counts per minute ⫾ SD. Significant suppression (p ⬍ .05) of the MLR by hUCMS cells is denoted by an asterisk for the blocking of responses at the highest dose of splenic fibroblasts that did not mediate significant suppression. This dose was 20,000 cells per well in (A) and 5,000 cells per well in (B). Abbreviations: MLR, mixed lymphocyte reaction; P, passage; UCMS, umbilical cord matrix stromal.

DISCUSSION

Figure 5. Suppression summary. The data from Figure 4 are summarized and expressed as percent age of suppression of the control mixed lymphocyte reaction response. The data used for this figure were derived from doses of cells added in which fibroblasts were not suppressive: 20,000 (isolates 4-3 and 4-5) and 5,000 (isolates 4-6 and 4-7) cells per well. Abbreviations: ND, not done; P, passage; UCMS, umbilical cord matrix stromal.

Focused Array Results As shown in supplemental online Fig. 1, cytokines implicated in hematopoietic stem cell proliferation, cell proliferation factors, or rescue functions or with specific cytokine activity were expressed by hUCMS cells. Specifically, mRNA for interleukins 1A, 1B, 6, 8, 11, and 14 was expressed by hUCMS cells. In addition, mRNA was present for BMP1, CSF3, FAM3C, GDF15, PDGFB, and tumor necrosis factors 4, 11b, 12, and vascular endothelial growth factor (VEGF) was expressed by hUCMS cells (supplemental online Fig. 1).

Here, the first description of the immune properties of Wharton’s jelly-derived cells (here called hUCMS cells) was provided. Five observations were reported. First, human UCMS cells suppressed the proliferation of stimulated immune cells. Con-A-stimulated rat splenocytes (xenograft model) or activated human peripheral blood mononuclear cells (allogeneic transplant model) were cocultured with human UCMS cells. Second, human UCMS cells did not stimulate proliferation of allogeneic or xenogeneic immune cells. Human UCMS cells were cocultured with two different isolates of purified T cells, and no proliferation of the T cells was found. When human UCMS cells were cocultured with rat splenocytes, no proliferation was observed. Third, RT-PCR indicated that hUCMS cells produced an immunosuppressive isoform of human leukocyte antigen (HLA), HLA-G6. Fourth, flow cytometry revealed that the expression of immune response-related surface antigens CD40, CD80, and CD86 was absent on hUCMS cells. Fifth, the presence of mRNA for cytokine genes by hUCMS cells was evaluated, and it indicated that hUCMS cells make several cytokines, such as IL-6 and VEGF. Together, these results indicate that human UCMS cells have immunosuppressive properties in vitro and that human UCMS cells have low immunogenicity. These findings indicate that UCMS cells that have been expanded for four to nine passages in vitro appear to have the same immune properties as MSCs derived from other tissue sources. Furthermore, these results suggest that UCMS cells would be tolerated in an allogeneic transplant.


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Figure 6. Reverse transcription polymerase chain reaction (RT-PCR) for HLA-G isoforms. RT-PCR for HLA-G5 and HLA-G6 isoforms revealed that human umbilical cord matrix stromal cells express mRNA for HLA-G6. Positive controls were human term placenta and jegHLA-G cells. Negative controls were JARp745 cells. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLA, human lymphocyte antigen; P, passage; UCM, umbilical cord matrix. Table 1. Flow cytometry results FACS antibody

CD40 CD80 CD86

hUCMS 21, passage 4

hUCMS 12, passage 4

hUCMS 9, passage 4

2% ND ND

ND ND ND

ND ND ND

Abbreviations: FACS, fluorescence-activated cell sorting; hUCMS, human umbilical cord matrix stromal; ND, not detected.

Properties of UCMS Cells The umbilical cord matrix, also known as Wharton’s jelly, is the loose connective tissue that surrounds and cushions the umbilical vessels. The Wharton’s jelly contains hyaluronic acid, collagen, and primitive multipotent cells [21, 24, 26, 28, 29, 36, 39, 40]. Human UCMS cells used here met the definition established for MSCs by the ISCT based upon their plastic adherence, morphology, surface phenotype, and multipotency (supplemental online Table 1). The differences between adult-derived MSCs and UCMS cells are that (a) UCMS cells have the potential for faster expansion ex vivo, and (b) UCMS cells have telomerase activity [22, 24, 28, 40], and the umbilical cord is relatively enriched for CFU-F compared with bone marrow [36]. Several laboratories have reported that 10,000 –15,000 nucleated cells can be isolated per centimeter of human umbilical cord length [24, 29, 34]; another laboratory has reported 106 nucleated cells per centimeter [36]. Thus, Wharton’s jelly is enriched for MSC-like cells. Furthermore, UCMS cells are www.StemCells.com

multipotent and can be differentiated into bone, cartilage, fat, and neural cells [28 –30, 40]. It is important to note that in vitro data must be confirmed with in vivo data. Although in vitro data appear to be fairly consistent among laboratories (showing suppression and low immunogenicity of MSCs), the animal data have been contradictory. Recently, it has been shown that porcine MSCs, which appeared to have low immunogenicity in vitro, elicited an immune response after intracardiac transplantation into allogeneic recipients, although subcutaneous transplants required a second injection to effect a similar reaction [41]. However, multiple injections of large numbers of allogeneic baboon MSCs (1 ⫻ 106 cells per kilogram of body weight) via multiple routes resulted in significant engraftment and the induction of host T-cell hyporesponsiveness, although all recipient baboons produced alloantibodies [42]. In contrast, it was reported that multiple injections of allogeneic swine UCMS cells could elicit an immune response [43]. This change is likely due to changes in major histocompatibility complex (MHC) class II expression, since human or swine UCMS cells could be induced to express MHC class II following a 48-hour exposure to interferon-␥ in culture [43]. Similarly, allogeneic swine UCMS cells activated by interferon-␥ exposure were immunogenic upon their first injection into swine, and nonactivated UCMS cells were not. These results do not discount the idea of using hUCMS cells in an allogeneic setting and suggest that care may be needed when multiple injections of UCMS cells are given. Work from several laboratories indicates that UCMS cells have therapeutic potential for neurodegenerative disease [24, 27], stroke [44], photoreceptor degeneration [40], and breast cancer [45]. Finally, UCMS cells may be useful for tissue engineering [25, 29, 46 – 49]. These factors together suggest that hUCMS cells may be an important source of cells for cell therapy in the future. The ability to use UCMS cells in allogeneic transplantation would enable their therapeutic use. For example, this would simplify the “stock” of cryogenically stored UCMS cells required for off-the-shelf therapeutic use.

Do MSCs Have Specific Immune Properties? In the Introduction, the minimal marker set of MSCs was given as plastic adherence, a particular surface marker set, and multipotency. MSCs may have additional properties. For example, it is well documented that (a) MSCs act as support cells for hematopoietic stem cells [50, 51], (b) they migrate to sites of pathology and infiltrate tumors [52, 53], and (c) they may have particular immune properties of low immunogenicity and immune suppression [2, 4, 5, 34]. A similar set of immune properties is reported here with UCMS cells. Furthermore, the current work suggests that the UCMS cells act via to suppress proliferation of activated splenocytes, specifically in allogeneic and xenogeneic formats. The suppression of the proliferation was 25%– 40% (in Fig. 1) to 38% (Fig. 2), showing good agreement among three methods used to evaluate xenogeneic suppression. The suppression in the allogeneic human MLR averaged 38% (range, 17%–54%; Fig. 5). UCMS cells did not inhibit proliferation of cultured splenocytes, and there is no evidence that UCMS cell coculture caused splenocyte death. Interestingly, a recent publication reports that VEGF and IL-6 secreted by MSCs result in immunosuppression [54]. VEGF is thought to cause this effect by blocking the emigration or differentiation of bone marrow lymphoid precursors [55]. IL-6 has been postulated to inhibit the differentiation of monocytes to dendritic cells [56]. The focused array data indicated that hUCMS cells express IL-6 and VEGF, as was reported previously [24]. The role of these factors will require confirmation to

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determine whether they are playing a role in the immune modulation observed here. MSCs have been reported in most publications to be MHC class II-negative (e.g., [1, 57]), but in at least two publications they have been reported as weakly positive [34, 58]. To date, cultured UCMS cells have been reported to be MHC class II-negative [28, 29, 36, 40]. As discussed above, exposure to interferon-␥ stimulates increased MHC class I and induces MHC class II expression by human and swine UCMS cells [43]. Importantly, UCMS cells express IL-6 and VEGF genes (supplemental online Table 1; [24]), and since, as stated above, these two proteins have recently been shown to be pivotal in the immunosuppressive capability of MSCs [54], their expression by the UCMS cells may be a mechanism for the immunosuppressive properties demonstrated here. In support of the cytokine gene expression profile provided here, the previous gene expression analysis of UCMS cells confirms the focus array data provided here [24], and Friedman et al. have reported that UCMS cells release hematopoietic and immune regulatory cytokines into the medium [59]. In addition, MSCs do not express costimulatory molecules (e.g., CD80, CD86, or CD40) that contribute to immunogenicity [34, 60]. Human UCMS cells do not express these molecules either (Table 1). Together, these findings may explain the lack of an apparent host immune response observed following transplantation of either porcine [21, 23] or human [24] UCMS cells into the rat brain, and they support the notion that UCMS cells share properties of MSCs.

Here, the one-way and two-way MLR assays were examined as a function of passage (time in cell culture). Thus, additional information was obtained. Specifically, the immunogenicity of human UCMS cells was low and did not change from passage 5 through passage 9 in four isolates examined. In contrast, human UCMS cells were found to be immunosuppressive at passages 5– 6 and passage 9, but the degree of immune suppression observed in two of three isolates may have decreased slightly during passage. Since chondrogenic differentiation alters the immunosuppressive properties of MSCs [65], it is possible that differentiation of UCMS cells may explain why two of the three isolates lost immunosuppressive properties at passage 9. This is not likely, though, because UCMS cells were not exposed to medium with specific differentiation factors, and there were no data to suggest that the UCMS cells were differentiating (e.g., no lipid in cells, no slowing down of proliferation, no evidence of matrix deposition, and no changes in cellular morphology to indicate differentiation of UCMS cells). Further work is needed to determine whether prolonged culture or, specifically, differentiation plays a role in UCMS cell immunophysiological properties. In this regard, recent work by Cho et al. indicates that culture of umbilical cord-derived cells with inflammatory cytokine interferon-␥ may increase immunogenicity by expression of MHC molecules [43]. The mechanism for this change in immune properties is unknown but may be due to changes in cytokine profile or nitric oxide release (Dr. L. Wang, Auburn University, personal communication).

HLA-G

Xenotransplantation Studies with hUCMS Cells

The reduced immunogenicity of MSCs has been likened to maternal acceptance of the fetal allograft [19]. The human MHC class I molecule HLA-G has long been known as a molecule selectively expressed by cytotrophoblastic cells. By inhibiting the cytolytic function of decidual natural killer cells, HLA-G protects the fetal tissue from the mother’s immune system. HLA-G molecules have 15 alleles [61– 63]; we tested for pan-HLA-G (data not shown) and for the soluble HLA isoforms HLA-G5 and HLA-G6. Since UCMS cells are fetal cells derived from postnatal tissue anatomically continuous with the placenta, we examined HLA-G expression in UCMS cells. The expression of pan-HLA-G (data not shown) by both early (P4) and later (P8) passage human UCMS cells and expression of the HLA-G6 isoform suggest that UCMS cells share some of the gene expression profile expressed by placental cells. Therefore, the importance of the presence of HLA-G in the UCMS cells in immune suppression and immunogenicity is currently unknown. A more complete examination of the role of HLA-G6 in the immune physiology of UCMS cells is slated for the future. An important physiological variable for cells under consideration for cellular therapy is how well those cells are tolerated by the host’s immune system. For example, will complete haplotype matching be required for engraftment, or will mismatched (e.g., allogeneic) transplantation be permitted? This is important information for two independent reasons. First, since graft rejection and graft-versus-host disease seen in bone marrow transplants are serious, life-threatening complications, it is important for engraftment, reduction of risk, and therapeutic efficacy to understand the immunogenicity and immune suppression function of therapeutic cells. Second, the tissue matching requirements will define commercial variables, such as how many samples the stem cell bank must contain to serve a diverse population. This mathematical exercise has been conducted by embryonic stem cell scientists, since embryonic stem cells will require tissue matching [64]. If allogeneic transplantation is safe and effective, a smaller bank of human UCMS cells would be required, compared with embryonic stem cells.

UCMS cells, unlike ESCs, do not induce tumors or death after 1 ⫻ 106 to 6 ⫻ 106 undifferentiated hUCMS cells were transplanted either i.v. or s.c. into Beige/SCID mice [24, 45]. In addition, UCMS cells are not acutely rejected when transplanted as xenografts in immune-competent rats. For example, pig UCMS cells undergo a moderated expansion following transplantation into rat brain without obvious untoward behavioral effects or host immune response [21, 23]. Pig UCMS cells were recovered from rat brain more than 6 weeks after transplantation without immune suppression. Similarly, human UCMS transplantation into rats did not trigger obvious host immune response around the transplantation site, although cells were not recovered more than 1 week after transplantation [24]. This degree of tolerance may be due to either immune property tested here: that is, either immunosuppressivity or low immunogenicity. In either case, the lack of infiltration by host immune cells bodes well for engraftment of UCMS cells. To date, there are no reports of engraftment, expansion, or differentiation of UCMS cells. Therefore, UCMS cells have not met the definition of true stem cells. It is possible that syngeneic grafting, perhaps together with opening of the niche via irradiation, will be needed to permit UCMS cells to engraft.

SUMMARY Human UCMS cells from passages 4 –9 have low immunogenicity and suppress the proliferation of activated immune cells. The mRNA for HLA-G6, an immunosuppressive HLA-class I marker synthesized by cytotrophoblasts at the maternal-fetal interface, was found in UCMS cells. In contrast, HLA-G5 expression was not found. This differentiates umbilical cord matrix stromal cells from cytotrophoblastic cells, which express both HLA-G5 and HLA-G6, and from bone marrow-derived MSCs, which do not express HLA-G. Human UCMS cells do not express the costimulatory surface antigens CD40, CD80, and CD86. Focused gene array data revealed that UCMS cells express IL-6 and

Weiss, Anderson, Medicetty et al. VEGF, molecules thought to play a role in MSC immune regulation. These data, taken together with previous observations following xenotransplantation of UCMS cells, suggest that hUCMS cells would be tolerated in allogenic transplantation.

ACKNOWLEDGMENTS We acknowledge the support of K-INBRE, Kansas State University (KSU) Developing Scholars program, KSU Department of Anatomy and Physiology, KSU College of Veterinary Medicine Dean’s office, KSU Provost’s office, KSU Terry C. Johnson Center for Basic Cancer Research, NIH (NS34160), and support from the State of Kansas to the Midwest Institute for Comparative Stem Cell Biology. The Northeast Kansas Parkinson Association is thanked for its generous support. We thank Robert Deans, Tony Ting, Kathy Mitchell, Alan Smith, Mahen-

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dra Rao, Sherry Flemming, and Frank Blecha for assistance with the research. We thank Drs. F. Blecha and S. Flemming for critically reviewing a draft of the manuscript. Dr. Susan Bennett and the OB/Gyn Staff at Mercy Medical Center are thanked for assistance in obtaining specimens. The anonymous donors are thanked for their contribution. S.M. is currently affiliated with Athersys Inc., Cleveland, OH.

DISCLOSURE

OF POTENTIAL OF INTEREST

CONFLICTS

M.L.W. is a paid consultant for the Regenerative Medicine Institute (Las Vegas, NV) and Toucan Capital Fund II (Bethesda, MD); K.R.M. is employed by Cognate Bioservices; S.M. is employed by Athersys; and M.L.W. has acted as a consultant and performed contract work for Toucan Capital Corporation.

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