In vitro immunologic properties of human umbilical cord perivascular cells

Cytotherapy (2008) Vol. 10, No. 2, 174
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In vitro immunologic properties of human umbilical cord perivascular cells J Ennis1, C Go¨therstro¨m2, K Le Blanc2,3 and JE Davies1 1

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada, and 2Division of Clinical Immunology, and 3Hematology Centre, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden

Background It has been shown recently that human umbilical cord perivascular cells (HUCPVC) are bio-equivalent to bone marrow-derived mesenchymal stromal cells (BM-MSC) in their mesenchymal differentiation and marker expression. HUCPVC populations provide high yields of rapidly proliferating mesenchymal progenitor cells. The question we wished to address, in two independent laboratory studies, was whether HUCPVC exhibit a similar in vitro immunologic phenotype to that of BM-MSC. Methods HUCPVC were isolated by physical extraction of umbilical vessels followed by enzymatic digestion of the perivascular cells, and lymphocytes were obtained from heparinized human peripheral blood. Experimental evaluations were lymphocyte proliferation in HUPCVC or BM-MSC co-cultures with peripheral blood lymphocytes (PBL), mixed lymphocyte cultures (MLC) containing BM-MSC or HUCPVC, CD25 and CD45 expression in co-cultures containing HUCPVC, and finally lymphocyte proliferation in TransWell MLC with HUCPVC.

Introduction The perivascular tissue of the human umbilical cord contains a rich source of mesenchymal precursor cells that we have called human umbilical cord perivascular cells (HUCPVC) [1]. In fact, the perivascular niche has recently been shown to be the source of mesenchymal progenitors in many organs [2] and bone marrow-derived multipotent mesenchymal stromal cells (BM-MSC; also known as mesenchymal stem cells) are themselves considered perivascular in origin [3]. The connective tissue matrix of the human umbilical cord has emerged as a promising cell source since McElreavey et al. [4] first

Results Both HUCPVC and BM-MSC showed no significant increase in proliferation of lymphocytes when co-cultured. The addition of 10% HUCPVC or BM-MSC significantly reduced proliferation of PBL in one-way MLC. Upon inclusion of HUCPVC with activated T-cell lines, the expression of both CD25 and CD45 showed a significant decrease. HUCPVC were able to reduce lymphocyte cell numbers significantly when separated with a membrane insert. Discussion HUCPVC are not alloreactive and exhibit immunosuppression in vitro. Lymphocyte activation is significantly reduced in the presence of HUCPVC, and the immunosuppressive effect of HUCPVC is due, in part, to a soluble factor. Thus HUCPVC shows a similar immunologic phenotype to BM-MSC. Keywords immunosuppression, mesenchymal stromal cells, perivascular, umbilical cord, MSC.

extracted fibroblast-like cells. Following this, cells have been extracted using several methodologies, resulting in populations capable of differentiating into neural [5
8], cartilage [9,10], muscle [11] and heart leaflet [12] cells. HUCPVC express the markers 3G5 and CD146, indicating their pericytic derivation, which is further demonstrated by the geographic exclusivity of these markers to the perivascular regions of the cord [13]. HUCPVC have been shown to be bio-equivalent to BM-MSC in both their marker expression profile (including a lack of telomerase) and their lineage differentiation capacity [1,14,15]. However, HUCPVC populations provide higher yields of

Correspondence to: J.E. Davies, Institute of Biomaterials and Biomedical Engineering, Room 407 Rosebrugh Building, 164 College Street, University of Toronto, Toronto, ON, Canada M5S3G9. E-mail: [email protected] – 2008 ISCT

DOI: 10.1080/14653240801891667

Immunologic properties of HUCPVC

rapidly proliferating colony-forming cells at harvest [14,16], which has enabled their designation as mesenchymal stromal stem cells based on single-cell seeded clonal self-renewal and multilineage differentiation assays [16]. In addition to the multilineage differentiation capacity of MSC, which makes them a powerful tool for tissue repair therapies, much recent attention has been focussed on their immunoregulatory functions. MSC, from human and other species, suppress many T, B and natural killer (NK) cell functions and may also affect dendritic cells (DC) [17,18]. Their intermediate expression levels of human leukocyte antigen (HLA) class I [19,20] and lack of expression of the co-stimulatory molecules CD80 and CD86 [21] may, individually or synergistically, play a role in them being poorly recognized by HLA-incompatible hosts and mitigating an active immune response [22]. These phenomena of immunoprivilege and immunomodulation/suppression have been demonstrated using in vitro assays, based upon mixed lymphocyte cultures (MLC) [20,23,24], in vivo experiments in several species [25
29] and human clinical applications [30,31]. Although BM-MSC are both immunoprivileged and immunomodulatory, which make them ideal for their use in HLA-mismatched allogeneic cell therapies, their harvesting for such therapies requires an invasive and elective procedure; and it is known that the number and maximal life span of BM-MSC declines with increasing age of the donor [32,33]. Thus alternative MSC sources, suitable for large-scale expansion for clinical applications, have been sought in other tissues, such as the stromal cells derived from adult dermolipectomies, which have also been shown to be immunomodulatory [34,35]. Because of their similarity to BM-MSC in both phenotype and differentiation, the question we wished to address, in two independent laboratory studies, was whether HUCPVC exhibit a similar in vitro immunologic phenotype to that of BM-MSC. We used different in vitro co-culture methodologies, but with the same outcome: HUCPVC do not induce proliferation of allogeneic lymphocytes but suppress alloreactivity in vitro.

Methods HUCPV cells Ethical consent for this research was obtained from the University of Toronto as well as Sunnybrook & Women’s College Health Sciences Centre. Umbilical cords were aseptically collected from Cesarean births of full-term

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babies, upon obtaining informed consent from the parent(s). The cords were immediately transported to the University of Toronto, where cells were extracted from the perivascular area under sterile conditions, as reported previously [1]. Briefly, 4-cm sections of cord were cut and the epithelium was removed. The vessels were then extracted, including their surrounding Wharton’s jelly, tied in a loop to reduce blood cell contamination, and digested overnight in a collagenase solution (Sigma, Oakville, ON, Canada). Upon removal from the digest the following day, the cells were rinsed in ammonium chloride (Sigma, ibid) to lyze any remaining red blood cells. Following this, the cells were plated out at a density of 4000 cells/cm2 in a-MEM containing 5% fetal bovine serum (FBS) and antimicrobials: penicillin G 167 U/mL, gentamicin 50 mg/mL and amphotericin B 0.3 mg/mL (Sigma, ibid). The cells were passaged when they reached 75
80% confluence, which was approximately every 6
7 days. Additionally, aliquots of cells were frozen in 10% DMSO (Sigma, ibid) and shipped using a vapor liquid nitrogen shipper (Chart SC 4/3V) to the Karolinska Institute for independent analyzes.

Lymphocytes White blood cells were extracted from heparinized blood taken from healthy donors. Cell separation was achieved by Ficoll
PaqueTM PLUS density gradient (Amersham Biosciences, Pittsburgh, PA, US), in which the cells were spun for 35 min at 380 g. The buffy coat was removed and counted using a ViCell-XRTM (Beckman Coulter, Mississauga, ON, Canada) with a protocol specific for lymphocytes, as determined by cell diameter and nucleus size. The cells were then plated out as per the requirements of the assay in RPMI-1640 media (Sigma, Oakville, ON, Canada) containing HEPES (25 mmol/L), L-glutamine (2 mmol/L), 10% FBS and antimicrobials (same constituents as HUCPVC).

Co-culture Triplicates of 1 104 HUCPVC were plated in 96-well plates (Falcon, Mississauga, ON, Canada). Once the cells had attached (approximately 2 h), 105 peripheral blood lymphocytes (PBL) were added to each well. The plates were incubated at 378C with 5% CO2 air in RPMI-1640 media containing HEPES (25mmol/L), L-glutamine (2 mmol/L), 10% FBS and antimicrobials. The cells were allowed to incubate for 6 days, after which they were

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stained with 5-bromo-2-deoxyuridine (BrdU; a base analog of thymidine) and measured using a Beckman Coulter FlowCenter. These results were confirmed by measurement of the lymphocyte proliferation by tritiated thymidine [3H] incorporation. PBL were purified from heparinized blood from healthy donors by Ficoll
Hypaque gradient (AxisShield PoC AS, Oslo, Norway) for 20 min at 600 g. Purified PBL were cultured in RPMI-1640 medium supplemented with HEPES (20 mmol/L), penicillin (100 U/mL), streptomycin (100 mg/mL), L-glutamine (20 mmol/L) (Gibco, Carlsbad, CA, US) and 10% heat-inactivated pooled human AB serum. Triplicates of 1 105 PBL from each donor were incubated at 378C in humidified 5% CO2 air on 96-well plates for 6 days (Nunclon, Copenhagen, Denmark), as described elsewhere [24]. Proliferation was measured after 24-hour tritium-labeled thymidine incorporation (1 mCi; Radiochemical Centre, Amersham, UK). On day 6, the cells were harvested automatically (Harvester 96; Tomtec, Orange, CT, US) on a glass fiber filter. Thymidine incorporation was expressed as the mean of triplicates in counts per minute (c.p.m.). In order to examine the response of resting lymphocytes to allogeneic HUCPVC or BM-MSC, HUCPVC/BM-MSC irradiated with 20 Gy were added to the cultures at a percentage of 0.01
10%.

One-way MLC In one-way MLC, PBL were isolated and purified, and responder PBL reacted against equal numbers of irradiated (20 Gy) stimulator PBL from a pool of donors in RPMI1640 medium supplemented as described above. Irradiated (20 Gy) third-party BM-MSC or HUCPVC were added in proportions ranging from 0.1% to 10% of responder cells. Tritiated thymidine was added 24 h before the end of the culture, and proliferation was measured on day 6, using the same methods as above.

and counted on a ViCell-XRTM cell counter. Two-way MLC were also performed in which the lymphocyte populations were separated from the HUCPVC by a 0.8-mm semi-permeable TransWell† membrane insert (Corning, Lowell, MA, US). The HUCPVC (1 104 cells/well) were cultured on the Transwell† surface and the insert was transferred to a 24-well plate containing PBL from two mismatched donors (as described above). The lymphocyte cell numbers were determined using the ViCell-XRTM on day 6.

Activated T-cell Line An activated T-cell line (ATL) was created using multiple doses of quiescent cells from one donor (treated with 20 mg/mL mitomycin C) to stimulate cells repeatedly from a mismatched donor. Upon the second stimulation, interleukin-2 (IL-2) was added (100 U/mL; BD Biosciences, Mississauga, ON, Canada) every 2 days with feeding. Thus an activated cell population with antibodies (Ab) to a specific donor was generated. Using these cells, the PBL co-culture and two-way MLC were carried out as described and assayed for cell proliferation and expression of CD25 and CD45.

Cellular staining In order to delineate between populations with greater accuracy, the ATL population was stained with PKH26 (Sigma, Oakville, ON, Canada). Cells were stained as per the protocol supplied with the product; the dye was added to the cell suspension for 2
5 min (2 10 6 molar PKH26 solution). After staining, the cells were suspended in supplemented media, spun down and washed. The cells were then included in a co-culture and measured for Ab expression (CD25, IL-2 receptor, marker of lymphocyte activation and CD45) gated on dye expression, thereby measuring only activated cells.

Statistics Two-way MLC Two-way MLC incorporate two proliferative lymphocyte populations from unmatched donors. This assay more closely mimics in vivo conditions, and was used to confirm the immunomodulatory capacity of HUCPVC. Briefly, 104 HUCPVC were allowed to attach to wells of a 96-well plate. Once these cells had attached (approximately 2 h), 105 cells of both lymphocyte populations were added to each well. The plates were allowed to incubate for 6 days,

Data sets were compared using ANOVA, followed by a post-hoc Tukey-HSD. P-values lower than 0.05 were considered significant.

Results HUCPVC do not induce allogeneic lymphocyte proliferation Upon addition of HUCPVC or BM-MSC at a dose range of 0.01
10% to a PBL population, there was no increase in

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40.894.8 103 cells), indicating their in vitro immunoprivilege in either resting or stimulated conditions (Figure 2).

HUCPVC reduce in vitro alloreactivity

Figure 1. HUCPVC and BM-MSC show no increase in the proliferation of resting lymphocytes, as determined by ANOVA, upon inclusion in a co-culture. Background (BG) proliferation has been set to 100% to reduce variation between experiments; BM-MSC n 5, HUCPVC n 4. This figure shows the average percentage proliferation (squares) and the range of data points (crosses).

proliferation of resting lymphocytes (Figure 1); in addition, the data were consistent with past work [24]. The proliferation of PBL after 6 days was recorded as incorporated radioactive thymidine and displayed as c.p.m., which was then set to 100% to reduce differences between experiments. This result was confirmed by flow cytometric measurement of BrdU incorporation (data not shown). HUCPVC were also analyzed for their effect on lymphocyte cell number upon inclusion in a co-culture with resting PBL or ATL. In both cases, HUCPVC caused no significant increase over control cell number (PBL, 35.293.1 103 cells; 10% HUCPVC, 45.095.7 103 cells; ATL, 38.8918.2103 cells; 10% HUCPVC,

Figure 2. HUCPVC do not increase resting or activated lymphocyte cell number. Addition of HUCPVC showed no significant increase in lymphocyte cell number compared with controls over 6 days in culture (n 6). This figure shows the average cell numbersstandard deviations.

In MLC, the addition of either HUCPVC or BM-MSC at the 10% dose level significantly reduced proliferation of PBL after 6 days, as measured by c.p.m., set relative to MLC-positive controls (MLC, 100%; 10% HUCPVC, 65.9913.9%; BM-MSC, 41.8927.3%), while none of the other concentrations exhibited a significant difference (Figure 3). Again, the data values reflected those we have published previously using BM-MSC [24]. HUCPVC were added after the start of a two-way MLC on days 3 and 5, and were able to reduce the lymphocyte cell number significantly. The cultures with HUCPVC added later were not significantly different from those added on day 0 (MLC, 18.690.9 103 cells; 10% HUCPVC day 0, 13.691.3 103 cells; day 3, 14.29 0.9 103 cells; day 5, 14.491.0 103 cells) (Figure 4), indicating that HUCPVC can reduce ongoing in vitro alloreactivity.

Reduction in activation ATL stained with PKH26 were added in a co-culture with 10% HUCPVC and assayed for their expression of CD25 (IL-2 receptor), a marker of lymphocyte activation, and CD45. Upon inclusion of HUCPVC, both the percentage of cells expressing CD25 (control, 100%; 10% HUCPVC,

Figure 3. HUCPVC reduce T-cell proliferation in one-way MLC. Positive control MLC proliferation has been set to 100% in the figure to reduce the variation in response between experiments. Addition of either 10% HUCPVC (n4) or BM-MSC (n6) significantly reduced proliferation, as determined by anova (P B0.05). The figure shows the average percentage proliferation (squares) and the range of data points (crosses).

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Figure 4. HUCPVC reduce lymphocyte cell number, even if added 3 and 5 days into the 6-day culture. Addition of HUCPVC showed a significant decrease in lymphocyte cell number compared with the control over 6 days in a two-way MLC. There was no significant difference among HUCPVC added on day 0, 3 or 5 (n 6). This figure shows the average cell numbersstandard deviations (*P B 0.05).

96.990.7%), and the mean fluorescence intensity (MFI) (control, 28.690.1; 10% HUCPVC, 3.7690.1) were significantly reduced (Figure 5). In addition, cells marked with PKH26 showed a significant reduction in CD45 (control, 100%; 10% HUCVPC, 99.690.1%; MFI control, 28.2094.24; 10% HUCPVC, 16.3391.27) (Figure 6).

HUCPVC act through a soluble factor Lymphocytes were cultured with HUCPVC and separated using a TransWell insert. The addition of 10% HUCPVC showed a significant reduction in lymphocyte cell number

Figure 5. HUCPVC reduce CD25 expression in ATL co-cultures. This figure illustrates both the average percentage expression (bar) and MFI (line) of CD25 expression on lymphocytes co-cultured with and without 10% HUCPVC. Averages are9standard deviations (n 3) (*P B0.05).

Figure 6. HUCPVC reduce CD45 expression in ATL co-cultures. This figure illustrates both the average percentage expression (bar) and MFI (line) of CD45 expression on lymphocytes co-cultured with and without 10% HUCPVC. Averages are9standard deviations (n 3) (*P B0.01).

over a 6-day culture period compared with the control (MLC, 40.7932.9 103 cells; 10% HUCPVC, 21.39 14.7 103 cells) (Figure 7). Soluble factors may therefore contribute to HUCPVC immunomodulation; however, what those factors are and how they affect lymphocytes is still unknown.

Discussion Our rationale for isolating the perivascular tissue as a source of MSC was predicated on two issues. First, the umbilical cord is a rapidly growing organ, achieving a length of 48
60 cm in only 40 weeks [36], with the majority of the growth occurring before 28 weeks but continuing to term [37]. Thus there must be a source of

Figure 7. HUCPVC act through a soluble factor. HUCPVC are able to significantly reduce lymphocyte cell number in MLC, when separated using a TransWell insert. The control (background; BG) lymphocyte cell number has been set to 100% in the figure to reduce the variation in counts between experiments. This figure shows the average percentage lymphocyte cell counts standard deviation (n 6) (*P B0.05).

Immunologic properties of HUCPVC

rapidly proliferating mesenchymal cells providing the stromal cells found in the extracellular connective tissue matrix. Secondly, the most likely place to locate these proliferating mesenchymal cells would be around the vasculature that is their source of nutrients. This perivascular tissue had been discarded in previous cell harvesting techniques [5,9,38]. To date, HUCPVC have been described as bio-equivalent to BM-MSC in differentiation, gene expression and marker expression profile [1,14]; the work here demonstrates their suitability for allogeneic use, as exemplified by in vitro methodologies established in the literature. With the finding that BM-MSC are able to evade allogeneic host immune recognition, the potential clinical applications of these cells have expanded enormously, as formerly they were primarily indicated for hematopoietic support [14] and differentiation into mesenchymal lineages for tissue engineering strategies [39]. Following encouraging in vitro results, largely conducted in MLC, early clinical studies with BM-MSC for the treatment of immune and inflammatory diseases have shown very promising results in rescuing patients from graft-versushost disease and Crohn’s disease [30,31,40]. The main limitation of BM-MSC, however, lies in their low frequency in adult marrow (1:100,000), which decreases with increasing donor age [32,33]. Thus other tissues in the body, such as adipose tissue harvested from lipo-aspirates [41] and cord blood, have been proposed as alternative sources of multipotential MSC. The limitation of the latter population is a low frequency of progenitors (1:2 108) and a low extraction success rate (29%) [32]. We have shown in this bilateral study that HUCPVC have a similar immunologic phenotype to BM-MSC, even in their dose-effect on lymphocyte proliferation (including the trend among doses). This correlates with previous work in which we showed the trend of BMMSC doses, although the cellular mechanisms underlying the dose
response have yet to be elucidated [24]. Indeed, the inability of HUCPVC to be recognized by HLAincompatible cells, and their ability to not only mitigate alloreactivity in a MLC but also decrease the level of lymphocyte activation, would indicate that the mechanism(s) of these immunologic effects in HUCPVC may be equivalent to those already described for BM-MSC. Interestingly, HUCPVC are able to reduce the proliferation of lymphocytes involved in an active immune

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response even if added after the immune response has begun. This has not been demonstrated previously with BM-MSC [24]. An unexpected result seen in the labeled ATL was the reduction of CD45 expression upon inclusion of HUCPVC. CD45 is expressed on all nucleated hematopoietic cells; however, a lack of expression is associated with immunodeficiency, as mutation in the CD45 gene has been implicated in severe combined immunodeficiency (SCID) [42]. Huntington & Tarlington [43] reviewed the data regarding expression of CD45 and its effect on immune responses, and found that CD45 is crucial for development of immune cells of different types (T, B and NK cells), while Dawes et al. [44] showed that both the levels of expression and combinations of CD45 isoforms play a critical role in lymphocyte function. However, the relationship between lower CD45 expression, as seen in our results, and immune responsiveness is far from simple, as McNeill et al. [45] have shown recently in mice that CD45 differentially regulates negatively and positively acting tyrosine kinase phosphorylation sites by a rheostat mechanism, to affect either reduced T-cell receptormediated signaling or hyperactivation of CD4  and CD8  T cells, and that maximal responsiveness is found with CD45 expression below normal expression levels. It is therefore not appropriate to speculate on the clinical relevance of our preliminary findings. We have isolated a population of cells equivalent to BMMSC in all known aspects of their functionality, while providing a source of mesenchymal cells with no foreseeable limitation of availability. While we recognize that further work is required to deconvolute the mechanisms by which these immunoregulatory actions affect individual cells of the immune lineages with both HUCPVC and BMMSC, our collective results in these independent studies indicate that HUCPVC represent a promising putative source of readily available MSC for cell-based therapies.

Acknowledgements The authors are grateful for financial support from the Ontario Research and Development Challenge Fund (ORDCF; grant to J. E. Davies) and the Natural Sciences and Engineering Research Council (NSERC; scholarship to J. Ennis). We would also like to thank Dr R. Moineddin for advice on statistical analyzes.

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