Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells

Experimental Hematology 32 (2004) 657–664

Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34⫹ cells Yi Zhanga, Changdong Lib,*, Xiaoxia Jianga, Shuangxi Zhanga, Ying Wua, Bing Liua, Peihsien Tanga, and Ning Maoa a Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, People’s Republic of China; Department of Gynecology and Obstetrics, Second Hospital of Jilin University, Changchun, People’s Republic of China

b

(Received 21 July 2003; revised 22 March 2004; accepted 2 April 2004)

Objective. Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34⫹ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34⫹ cells from UCB in vitro. Materials and Methods. Human placenta MPC was isolated and characterized by morphologic, immunophenotypical, and functional analysis. UCB CD34⫹ cells were expanded by coculture with placental MPC. Suitable aliquots of cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay. Results. In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin, and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and a-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes. In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of cytokines, was significantly enhanced: CD34⫹ cells by 14.89 ⫾ 2.32 fold; colonyforming cell (CFC) by 36.73 ⫾ 5.79 fold; and LTC-IC by 7.43 ⫾ 2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli. Conclusion. These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro. 쑖 2004 International Society for Experimental Hematology. Published by Elsevier Inc.

Human umbilical cord blood (UCB) is an attractive source of hematopoietic stem cell (HSC) transplantation in children. Despite its higher ratio of hematopoietic progenitors than those from bone marrow (BM) and mobilized peripheral blood (PB), the lower total number of progenitors in UCB has severely restricted its widespread clinical use in adults [1,2]. To overcome this problem, two categories have been applied to ex vivo expansion of HSC within UCB. Various combinations of cytokines can significantly expand the total number of hematopoietic cells at the cost of HSC differentiation, which can be largely prevented by coculture with stromal cells instead.

Offprint requests to: Ning Mao, M.D., Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, China; E-mail: [email protected] *Dr. Zhang and Dr. Li contributed equally to this work.

0301-472X/04 $–see front matter. Copyright doi: 1 0. 10 1 6 / j .e x p he m.2 0 04 .0 4 .0 0 1

Mesenchymal progenitor cell (MPC) is multipotent progenitor that can self-renew and terminally differentiate into multiple lineages, including bone, adipose, cartilage, nerve, muscle, and BM stromal cells either in vitro [3–6] or in vivo [7–10], thus indicating its great therapeutic potential. As has been reported, MPC has been isolated from several mesenchymal tissues, such as BM, muscle, bone, cartilage, and tendon [11]. As the main source for both experimental and clinical studies, particularly HSC transplantation, BM-derived MPC has been shown to enhance engraftment and reduce severity of graft-vs-host disease [12– 14]. However, the decrease of BM MPC with age necessitates the search for alternative sources of MPC having the capacity to support hematopoiesis. Human placenta can secrete hematopoietic growth factors stimulating hematopoietic colony formation [15]. Hence, in a simple scenario, the placenta may be used as s “feeder

쑖 2004 International Society for Experimental Hematology. Published by Elsevier Inc.

658

Y. Zhang et al. / Experimental Hematology 32 (2004) 657–664

layer” suitable for expansion of primitive hematopoietic progenitors within UCB that has been concomitantly collected. In the present study, we show that MPC can be isolated from human placenta, having differentiating and immunoregulatory potential equivalent to those from BM, and more importantly is capable of expanding long-term culture initiating cell (LTC-IC) from UCB CD34⫹ cells in synergy with cytokines in vitro.

Materials and methods Isolation and culture of human placental adherent cells UCB and fetal placenta were collected from normal full-term pregnancies according to the regulations of the Research Ethics Committee of North Taipinglu Hospital, Beijing. Loose chorion, amniotic sac, and decidua were removed from the placenta. The placenta was washed extensively with phosphate-buffered saline (PBS) and flushed with perfusate and Iscove’s modified Dulbecco medium supplemented with heparin 12.5 U/mL, penicillin 50 U/mL, and streptomycin 50 mg/mL through the arterial-vein circuit to eliminate tissue residual blood. The placenta was soaked with 200 to 250 mL of medium for 12 to 24 hours at 20⬚ to 25⬚C. The mononuclear cells in the medium were recovered by Percoll density gradient fractionation (⬍1.073g/mL, Pharmacia, Piscataway, NJ, USA), washed, and resuspended in Dulbecco’s modified Eagle medium-low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS; Stem Cell Technologies, Vancouver, BC, Canada). After 10 days of culture with 5% CO2 at 37⬚C, individual colonies were isolated and expanded. When the cultures reached approximately 90% monolayer confluence, cells were recovered by using 0.25% trypsin-1 mM EDTA for culture expansion and further identification. Characterization of culture-expanded human placental adherent cells Placental adherent cells culture expanded from a single colony were phenotypically characterized by flow cytometry (BectonDickinson, San Jose, CA, USA; n ⫽ 6). Fluorescein isothiocyanateconjugated or phycoerythrin-conjugated antibodies against CD14, CD29, CD31, CD34, CD44, HLA-ABC, HLA-DR, CD73, CD166 (PharMingen, San Diego, CA, USA); CD45 (Becton-Dickinson); and CD105 (SEROTEC, Oxford, UK) were used. Moreover, human placental adherent cells were analyzed by immunocytochemistry. Cells were fixed in equal volumes of methanol and acetone for 1 minute at room temperature, washed with PBS, incubated with 3% hydrogen peroxide (H2O2; Sigma-Aldrich, St. Louis, MO, USA), and blocked with 10% normal goat serum at room temperature. Washed cells were incubated with the following primary antibodies: rabbit anti-human laminin polyclonal antibody (Dako, Gostrup, Denmark), mouse anti–α-smooth muscle actin mAb (Sigma-Aldrich), rabbit anti-human fibronectin polyclonal antibody (Sigma-Aldrich), and mouse anti-vimentin McAb (Sigma-Aldrich) at 1:100 dilutions with antibody diluents (Dako) for 30 minutes at room temperature. Anti-mouse or anti-rabbit peroxidase-conjugated immunoglobulin G antibody (Santa Cruz, Santa Cruz, CA, USA) was used as a secondary antibody at 1:100 dilution, and 3,3′-diaminobenzidine in chromogen solution (Dako) was applied to the slides, which were counterstained with 0.1% Mayer hematoxylin solution (Sigma-Aldrich).

To study adipogenic differentiation potential, cells (at passages 3–10) were incubated with DMEM-high glucose (HG) plus 10% FBS supplemented with 10⫺3 mM dexamethasone, 0.5 mM isobutyl methylxanthine, 0.2 mM indomethacin, and 10 µg/mL insulin (Sigma) for 2 weeks. To demonstrate the presence of adipocytes, cytoplasmic inclusions of neutral lipids were stained with oil-red-O (Sigma). Osteogenic differentiation was assessed by incubating the cells with DMEM-HG with 10% FBS supplemented with 10⫺4 mM dexamethasone, 0.5 mM ascorbic acid, and 10 mM β-glycerol phosphate (Sigma) for 2 to 4 weeks. Osteoblasts were identified by immunocytochemical stain with alkaline phosphatase. To assess mineralization, deposit calcium in cultures was stained with silver nitrate (Sigma) by the method of von Kossa. Medium with DMEM containing 2.5% FBS, 50 ng/mL transforming growth factor-β1 (Peprotech, London, UK), 50 µg/mL ascorbic acid, 1 mM sodium pyruvate, 6.25 µg/mL bovine insulin, 6.25 µg/mL transferrin, 6.25 µg/mL selenious acid, and 1.25 µg/mL bovine serum albumin was used for chondrogenic differentiation. Alcian blue staining was used to assess the formation of extracellular matrix, which is the mark of chondrogenic differentiation. Coculture of human placental MPC and UCB CD34⫹ cells Mononuclear cells were isolated from UCB using Ficoll Hypaque (density 1.077, Sigma) density centrifugation. The CD34⫹ fraction in MNC was directly isolated with anti-CD34 antibodies (QBEND/ 10 mouse immunoglobulin G; Miltenyi Biotec, Bergisch Gladbach, Germany) conjugated with microbeads and miniMACS column. The efficiency of the purification was verified by flow cytometry counterstaining with phycoerythrin–anti-CD34 antibody. After γirradiation at a dose of 12Gy, 2.0 × 105 monolayer-derived adherent cells from human placenta were seeded in 24-well plates (Costar, Bethesda, MD, USA). At 24 hours, 1 × 104 cord blood CD34⫹ cells were plated in long-term culture (LTC) medium (Myelocult H5100; Stem Cell Technologies) supplemented with 10⫺6 M hydrocortisone (Stem Cell Technologies). The cells were expanded for 12 days under three conditions: 1) cytokines alone (Flt ligand [FL] 50 ng/mL, stem cell factor [SCF] 50 ng/mL, thrombopoietin (TPO) 20 ng/mL, Peprotech); 2) MPC alone; or 3) cytokines plus MPC. At 12 days, nonadherent cells were collected and assayed as follows. 1) CD34 analysis by flow cytometry; 2) Nonadherent cells were grown in triplicate cultures in methylcellulose containing 50 ng/mL SCF, 10 ng/mL interleukin-3 (IL-3), 10 ng/mL GMCSF, 10 ng/mL G-CSF (Peprotech), and 4 U/mL erythropoietin (EPO) (Kirin, Tokyo, Japan), and incubated at 37⬚C with 5% CO2 for 12 to 14 days. Colonies with greater than 50 cells were designated as CFC. 3) For LTC-IC analysis, cells at dilutions of 100, 300, 900, 2700, and 8100 cells were plated at 24 wells each into 96-well plates containing LTC medium supplemented with 10⫺6 M hydrocortisone and a nearly confluent monolayer of irradiated allogeneic human BM stroma and maintained at 37⬚C and 5% CO2. Weekly half-media changes were performed. After 5 weeks, both nonadherent and adherent cells were transferred from the 96-well plates on a well/well basis into fresh 96-well plates with complete methylcellulose and cultured as described for CFC. A well that generated at least one progenitor colony was considered positive for limiting dilution calculations of LTC-IC frequency. LTC-IC frequency was estimated using Poisson statistics [16]. Absolute LTC-IC counts were calculated by multiplying the total nucleated cell number measured at 0 or 12 days with LTC-IC frequency

Y. Zhang et al. / Experimental Hematology 32 (2004) 657–664

measured at several time points. Control experiments were performed by culturing UCB CD34⫹ cells either in the absence of a feeder layer or over BM-derived MPC. Cytokine expression of human placental MPC detected by reverse transcriptase polymerase chain reaction Total RNA was extracted from cultured cells using Trizol reagent (Gibco Laboratories, Carlsbad, CA, USA), following the manufacturer’s instructions. RNA was converted to cDNA using oligo-dT primer and AMV reverse transcriptase (RT; Takara, Kyoto, Japan). Polymerase chain reaction (PCR) cycles were optimized for each set of primers (Table 1). The PCR products were analyzed by electrophoresis on 1.5% agarose gel containing 1µg/mL ethidium bromide. Mixed lymphocyte reactions PB mononuclear cells were isolated from heparinized blood by density gradient centrifugation. Human CD3⫹ T lymphocytes were isolated from PB mononuclear cells by a miniMACS magnetic cell sorting system (Miltenyi Biotec). UCB T lymphocytes were obtained from CD34⫺ cells by isolation of CD34⫹ cells with the miniMACS system. Cell counts and viability were assessed by trypan blue dye exclusion. Responding cells (2 × 105 per well), and an equal number of irradiated (30 Gy) stimulating cells were mixed at different ratios with MPC in 96-well culture plates to ensure efficient cell–cell contact for 4 days with 1µCi per well 3 H-thymidine(3H-TdR) pulsed during the last 16 hours in 0.2 mL RPMI 1640 medium (GIBCO BRL) containing 20% heat-inactivated FBS. T-cell proliferation was evaluated by measuring 3HTdR using a liquid scintillation counter. The experiments were repeated at least three times.

659

Statistical analysis Results are expressed as mean ⫾ SD. Statistical comparisons were performed using the two-sided Student’s t-test.

Results Morphology and growth characteristics of human placental adherent cells Human placental nucleated cells separated by density gradient were cultured at low density (12,000/cm2). After 10 days of inoculation, formed individual colonies displaying fibroblast-like morphology (Fig. 1A and B). These adherent cells could be readily expanded in vitro by successive cycles of trypsinization, seeding, and culture every 3 days for 20 passages without visible morphologic alteration (Fig. 1C–F). The growth kinetics of single colony-derived adherent cells was measured at passage 3. The mean cumulative time of population doublings was 21.25 hours. Immunophenotypic characterization of human placental adherent cells The immunophenotype of the human placental adherent cells was determined by flow cytometry and in situ immunocytochemistry. The culture-expanded adherent cells were stained positive for CD29, CD44, CD73 (SH3, SH4), CD105 (SH2), CD166, HLA-ABC, laminin, fibronectin, and vimentin but

Table 1. Primers used to detect cytokines and lineage-specific marker genes in normal or induced human placental mesenchymal progenitor cells Primer sequence

T (⬚C)*

Size (bp)

Reference no.

5′-CTC GCG CTA CTC TCT CTC TCT TTC TGG-3′ 5′-GCT TAC ATG TCT CGA TCC CAC TTAA-3′ 5′-AAC ACA CTT AAA GCA GCC AC-3′ 5′-TTT ACA GAA CGC ATC AGC AA-3′ 5′-GTAGCCGCCCCACACAGACAGCC-3′ 5′-GCCATCTTTGGAAGGTTCAGG-3′ 5′-TTGGGAGTGGACACCTGCAGTCT-3′ 5′-CCTTGGTGAAGCAGCTCTTCAGCC-3′ 5′-GTCTCCTGAACCTGAGATGAGACA-3′ 5′-AAGGGGATGACAAGCAGAAAGTCC-3′ 5′-AGCTTCCTGCTCAAGTGCTTAGAG-3′ 5′-TTCTTCCATCTGCTGCCAGATGGT-3′ 5′-TGGAGCCCAACAACCTATCTC-3′ 5′-GGGCTGAAAGGCACAATTTGGT-3′ 5′-CTCCTATTTAATCCTCTCGTC-3′ 5′-TACTACCATCTCGCTTATCCA-3′ 5′-GTGCCATACCAGTTAAACA-3′ 5′-CTTACTTGGAAGGGTCTCT-3′ 5′-TGTCAGTACTGTCGGTTTC-3′ 5′-AATGGTGATTTGTCTGTTG-3 5′-AGTGGAGACTACTGGATTGA-3′ 5′-AGTGTACGTGAACCTGCTAT-3′ 5′-TTACAGATCTCCATTTATTGC-3′ 3′-TTCATCTCACTCCCAGACT-5′

58

335

6

55

129

SD

58

174

15

57

248

15

55

286

15

55

346

15

58

333

15

58

177

15

55

169

SD

55

241

SD

60

394

SD

60

479

SD

Gene β2-Microglobulin IL-3 IL-6 M-CSF GM-CSF G-CSF FL SCF Osteopontin PPARγ2 Collagen II KDR

*Annealing temperature. SD ⫽ self-designed primer sequence.

660

Y. Zhang et al. / Experimental Hematology 32 (2004) 657–664

Figure 1. Morphology of human placenta-derived adherent cells in culture. Nucleated cells isolated from placenta became adherent after 48 hours of plating (A) and after 10 days formed an individual colony displaying a fibroblast-like morphology (B). The adherent cells at three passages (C–D) were stained with Wright-Giemsa (E–F). Original magnifications: C, E ×40; D, F ×100.

were negative for CD14, CD34, CD45, HLA-DR, α-smooth muscle actin, which is generally consistent with BM MPC (Fig. 2 and data not shown). The absence of CD31 and KDR expression ruled out the possibility of contamination of endothelial cells (Fig. 3). The adherent cells isolated from six independent colonies showed a similar phenotype spectrum even beyond passage 15. In addition, cryopreservation did not alter these immunophenotypic features. Human placental adherent cells were capable of differentiating into adipocytes, osteocytes, and chondrocytes Adipogenic differentiation of placental adherent cells was apparent after 3 to 5 days of induction, indicated by accumulation of oil-red-O staining lipid-rich vesicles. The lipid

vacuoles continued to develop over time, coalesced, and eventually filled the cell (Fig. 4A–C). Under osteogenic conditions for 2 weeks, they also could form aggregates or nodules displaying alkaline phosphatase activity. Calcium deposits, evaluated by the von Kossa method, were evident at week 3 (Fig. 4D–F). Finally, formation of type II collagen in the extracellular matrix of induced cells could be detected by Alcian blue staining after 3 weeks of culture in chondrogenic medium (Fig. 4G–I). In agreement with the results of differentiation assays, RT-PCR also demonstrated that the adherent cells displayed corresponding transcriptional expression of paroxysm proliferation activated receptor gamma 2 (PPARγ2), osteopontin (OP), and type II collagen under specific adipogenic,

Figure 2. Immunophenotyping of culture-expanded adherent cells from human placenta. Flow cytometric analysis of the status of mesenchymal, endothelial, and hematopoietic cell surface markers as well as adhesion-related antigens on the human placental single colony-derived adherent cells.

Y. Zhang et al. / Experimental Hematology 32 (2004) 657–664

661

Figure 3. Absence of endothelial specific marker CD31 and KDR in human placental culture-expanded adherent cells. Endothelial cell surface marker CD31 was not detected in the placental culture-expanded adherent cells (B) by flow cytometry with the human umbilical vein endothelial cells (A) as a positive control. Furthermore, RT-PCR demonstrated the absence of KDR (479 bp) expression in placental culture-expanded adherent cells (C, lane 2), while its mRNA was found in umbilical vein endothelial cells (C, lane 3). β2-Microglobulin (335 bp) served as an internal control. DNA marker is shown in lane 1.

osteogenic, and chondrogenic inductive cultures, respectively (Fig. 5). The morphologic, immunophenotypic, and differentiation assays described strongly indicated that the adherent cells isolated from human placenta were MPC resembling those from other sources. Human placental MPC could expand hematopoietic progenitors within UCB in vitro To determine whether placental MPC was capable of supporting the expansion of UCB-derived hematopoietic cells,

Figure 4. Induced differentiation of placental MPC into adipocytes, osteocytes, and chondrocytes. Adipogenesis was indicated by the presence of neutral lipid vacuoles that stain with oil red O (B–C and A show nonadipogenesis). Accumulation of intracytoplasmic alkaline phosphatase and calcium oxalates with von Kossa staining shows differentiation of MPC into the osteoblast lineage (E–F and D show controls). Chondrogenic differentiation was indicated by Alcian blue staining to detect type II collagen (H– I and G show controls).

placental MPC (n ⫽ 3) was irradiated and cocultured with CD34⫹ cells from UCB (1 × 104cells per well) in LTC medium. As control, CD34⫹ cells were overlaid on wells with BM MPC and/or cytokine cocktails. The total number of mononuclear cells, CD34⫹ cells, CFC, and LTC-IC was evaluated after 12 days of inoculation. Consistent with previous reports, the expansion magnitude of total nucleated cells, CD34⫹ cells, and CFC (Table 2) by placental MPC alone (23.09 ⫾ 3.44 fold, 4.85 ⫾ 0.72 fold, and 11.46 ⫾ 1.72 fold) or BM MPC alone (17.81 ⫾ 3.48 fold, 3.35 ⫾ 0.65 fold, and 7.74 ⫾ 1.50 fold ) was far lower than that by cytokines stimulation (55.92 ⫾ 9.54 fold, 5.34 ⫾ 0.91 fold, and 17.63 ⫾ 3.05 fold, p ⬍ 0.01) (Table 2). However, the combined use of placental MPC with additive cytokines manifested the most potent expansion capacity (p ⬍ 0.01) among the five groups, with the number of total nucleated cells increasing by 105.92 ⫾ 16.55 fold, CD34⫹ cells by 14.89 ⫾ 2.32 fold, and CFC by 36.73 ⫾ 5.79 fold, respectively (Table 2).

Figure 5. RT-PCR analysis for expression of differentiation marker genes. Loading of PCR products of β2-microglobulin served as internal control. In adipogenic, osteogenic, and chondrogenic differentiation conditions, PPARγ2 gene (241 bp) was detected 7 days after induction. Osteopontin and type II collagen genes (169, 394 bp) were detected 10 days after induction.

662

Y. Zhang et al. / Experimental Hematology 32 (2004) 657–664

Table 2. Fold expansion of CD34⫹ cells in the presence of mesenchymal progenitor cells derived from placenta and bone marrow at 12 days of culture

C BM-MPC P-MPC BM-MPC ⫹ C P-MPC ⫹ C

Total nucleated cells

CD34⫹ cells

CFC

LTC-IC

55.92 ⫾ 9.54 17.81 ⫾ 3.48 23.09 ⫾ 3.44 65.25 ⫾ 9.77 105.92 ⫾ 16.55*

5.34 ⫾ 0.91 3.35 ⫾ 0.65 4.85 ⫾ 0.72 7.46 ⫾ 1.11 14.89 ⫾ 2.32*

17.63 ⫾ 3.05 7.74 ⫾ 1.50 11.46 ⫾ 1.72 22.27 ⫾ 3.32 36.73 ⫾ 5.79*

3.54 ⫾ 1.50 1.66 ⫾ 0.56 2.16 ⫾ 0.55 4.63 ⫾ 1.60 7.43 ⫾ 2.66*

Values indicate the fold increase compared with the initial number of cells. Results are given as mean ⫾ SD from one representative experiment; the other two experiments demonstrated similar results. *p ⬍ 0.05, calculated by the Student’s t-test, when comparing data of the P-MPC⫹C group with data in the other groups. BM-MPC ⫽ bone marrow-derived mesenchymal progenitor cell (MPC) without cytokines; BM-MPC⫹C ⫽ bone marrow-derived MPC with cytokines; C ⫽ cytokines, including SCF 50 ng/mL, FL 50 ng/mL, and TPO 20 ng/mL; P-MPC ⫽ placental MPC without cytokines; P-MPC⫹C ⫽ placental MPC with cytokines.

LTC-IC assay, the quantitative definition of HSC proliferation in vitro, was further explored to determine whether placental MPC could support the expansion of UCB-derived primitive hematopoietic cells. As shown in Table 2, the yields of LTC-IC expansion in the presence of cytokines only (3.54 ⫾ 1.50 fold), although much higher than placenta MPC (2.16 ⫾ 0.55 fold) and BM MPC (1.66 ⫾ 0.56 fold), was lower than BM MPC plus cytokines (4.63 ⫾ 1.60 fold, p ⬍ 0.05). The placental MPC plus cytokines demonstrated the most powerful activity (7.43 ⫾ 2.66 fold, p ⬍ 0.01). This remarkable amplification might result from expansion of total cells, with similar LTC-IC frequency among all groups. These findings suggest that the placental MPC plus cytokines might be a novel as well as efficient feeder layer for UCB-derived hematopoietic progenitor cells. RT-PCR assay showed that human placental MPC expressed SCF, FL, IL-6, and M-CSF. Exposure of these cells to IL-1α and TNF-α could induce the expression of GCSF and GM-CSF (Fig. 6). β2-Microglobulin mRNA was amplified in the analysis in order to monitor equal input of RNA into the reactions. Immunosuppressive effects of human placental MPC in vitro To test the immunoregulatory effect of placental MPC, T lymphocytes (2 × 105) isolated from PB or UCB, cocultured for 4 days with 2 × 105 irradiated allogeneic lymphocytes as stimulators, showed a strong proliferation response (mean incorporation of 5577.2 ⫾ 654.91 cpm in PB; 6426.0 ⫾ 304.45 cpm in UCB) (Fig. 7). This could be suppressed in a dose-dependent way by the addition of increasing numbers of placental MPC to the cultures, resulting in a significantly lower response when 8 × 104 MPC was added to the culture system (mean incorporation of 1019.2 ⫾ 493.25 cpm in PB; 2096.67 ⫾ 160.31 cpm in UCB, p ⬍ 0.01) and a lower response when MPC was 2.5 × 103 (mean incorporation of 3525.21 ⫾ 784.43 cpm in PB; 5150.67 ⫾ 532.92 cpm in UCB, p ⬍ 0.01) (Fig. 7A). As shown in Figure 7B, when lymphocytes were cultured with a third-party MPC, a similar

suppressive effect was detected. The data indicated that placental MPC, whether from the same source as the stimulator or from a third party, was able to inhibit allogenic lymphocytes proliferation. To determine whether inhibition of T-lymphocyte proliferation was caused by coculture with MPC or was a bulk effect, we cultured T lymphocytes with irradiated allogeneic human umbilical vein endothelial cells. Under these culture

Figure 6. Gene expression of cytokines by human placental MPC in culture. Total RNA was isolated from placental MPC cultured in MPC medium or MPC medium with IL-1α (10 U/mL) or TNF-α (40 ng/mL) for 24 hours. Total RNA samples were used for synthesis of cDNA, which then was amplified for 35 cycles using specific primers for cytokines genes. Amplified products were analyzed on 1.5% agarose gels and stained with ethidium bromide. To monitor equal input of RNA in the reverse transcriptase reactions, β2microglobulin gene amplification was used.

Y. Zhang et al. / Experimental Hematology 32 (2004) 657–664

Figure 7. Human placental MPC decreased the proliferative response of allogeneic T cells from either peripheral blood (PB) or umbilical cord blood (UCB) when cocultured. Lymphocytes from PB or UCB were cocultured with various doses of irradiated allogeneic (A) or third-party (B) placental MPC. Proliferative activity occurred in a dose-dependent fashion. Bars show nonMPCs (0), responders ⫹ (0.25 × 104) MPCs, responders ⫹ (0.5 × 104) MPCs, responders ⫹ (1 × 104) MPCs, responders ⫹ (2 × 104) MPCs, responders ⫹ (4 × 104) MPCs, and responders ⫹ (8 × 104) MPCs. Responder cells were 2 × 105 peripheral blood lymphocytes or UCB T cells. Data are expressed as mean ⫾ SD of triplicates of three separate experiments. Significance was determined using the Student’s t-test. Human vein endothelial cells (VEC) were used as the control group.

conditions, no suppression of T-lymphocyte proliferation was detected (Fig. 7).

Discussion MPC has the capability for self-renewal and differentiation into various lineages of mesenchymal tissues. These features of MPC attract much attention from investigators with regard to tissue engineering and cell-based therapies. Despite the fact that BM represents the main available source of MPC, the use of BM-derived cells is not always acceptable because of the significant decrease in cell number and proliferation/ differentiation capacity with age. Placenta is likely a feasible source of MPC for two reasons. First, human placenta, as a castoff, can be obtained easily. Second, during ontogeny, the placenta, which contains a great deal of mesenchyma,

663

is formed from extraembryonic mesoderm [17]; thus, MPC can be found in placenta. Our results confirmed this assumption. In this study, we were the first to successfully isolate and identify human placental MPC, which was easily culture expanded and harbored similar morphologic, immunophenotypic, and differentiation characteristics to BM MPC. Placental MPC was clearly nonhematopoietic and nonendothelial; it was consistently negative for CD14, CD31, CD34, and CD45 and had no KDR expression. Like BM MPC, it expressed some mesenchymal markers, such as CD73 and CD105. In addition, it could differentiate into at least three different tissues: fat, bone, and cartilage. These cells were not isolated from UCB of the same donor, thus emphasizing that placental MPC did not originate from the circulation. The main obstacle to UCB transplantation in adult recipients is the insufficiency of hematopoietic progenitors. Hence, in this investigation we explored placental MPC as the feeder layer of UCB CD34⫹ cells and found that it could, in synergy with extra cytokines, dramatically expand CD34⫹ cells by 14.89 ⫾ 2.32 fold , CFC by 36.73 ⫾ 5.79 fold, and LTCIC by 7.43 ⫾ 2.66 fold over a 12-day period. Such a placental stromal /UCB in vitro expansion system may be illustrated, at least in part, by recent findings on placenta. In 1979, the mouse placenta was shown to harbor B-cell progenitors [18], and its hematopoiesis capacity has recently been fully reported [19–22]. As reported, the mouse placenta initiates hematopoiesis at the stage of 15 somite pairs (sp), and the frequency and proliferative potential of early hematopoietic progenitors within this organ are strikingly similar to those in human UCB rather than fetal liver and yolk sac. The authors pinpoint that such similarities, together with the anatomic and developmental relationships between umbilical cord and placenta, reveal the possibility that cord blood progenitors come from the placenta [23]. Thus, it comes as no surprise that the MPC isolated from human placenta was able to efficiently amplify the CFC together with LTC-IC pool of UCB in vitro. However, further in vivo data are required to confirm its hematopoiesis-supporting function and clinical application. Beyond that, we assessed the immunomodulatory capacity of placental MPC, which is important for clinic application. Animal models and in vitro studies showed that MPC is not substantially immunogenic and may actually inhibit both primary and secondary mixed lymphocyte reactions (MLR). Ongoing MLR may be suppressed by adding the responder, stimulator, or, intriguingly, third-party MPC [24– 27], correlating well with the peculiar immunophenotypic features of MPC, such as the lack of HLA class II and T-cell costimulatory molecule B7 [28,29]. Moreover, an ongoing clinical trial has provided further exciting insight into the function of MPC. Lazarus et al. [30] reported a phase I study of MPC dose escalation that evaluated the cotransplantation of HLA-identical MPC together with either PB- or BMderived hematopoietic stem cells from the same donor in

664

Y. Zhang et al. / Experimental Hematology 32 (2004) 657–664

patients with advanced hematologic malignancies. The patients who received coinfusion of allo-MPC along with alloBM were observed to have no significant toxicities and experienced a reduction of acute and chronic graft-vs-host disease [30]. Our result demonstrated that 1) culture-expanded placental MPC had an inhibitory effect on T-cell proliferation triggered by allogeneic PB- and UCB-derived T lymphocytes; and 2) this effect was dose dependent and still evident when MPC was from a third party. Although preliminary, this observation provides additional evidence on the potential immunomodulatory properties of human placental MPC, which may play a major role in the induction of tolerance to allogeneic, especially UCB transplantation. The mechanism underlying placental MPC-mediated suppression of T-cell proliferation requires further investigation using appropriate in vivo models. In conclusion, isolation of MPC from human placenta is promising not only in terms of origin but also as a novel microenvironment system with hematopoietic supportive and immunoregulatory features.

Acknowledgments This study was supported by a grant from National Natural Science Foundation (No. 30271245), the National “863” Program (No. 2001AA216141 and 2003AA205170), and National “973” Program (No.G1999054302). The authors thank Dr. Gensheng Feng for critical review of the manuscript.

References 1. Broxmeyer HE, Douglas GW, Hangoc G, et al. Human UCB as a potential source of transplantable stem/progenitor cells. Proc Natl Acad Sci U S A. 1989;86:3828–3832. 2. Cairo MS, Wagner JE. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood. 1997;90:4665–4678. 3. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical use. Exp Hematol. 2000;28:875–884. 4. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol. 1999;181:67–73. 5. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–147. 6. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 2002;105:93–98. 7. Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol. 2002;174:11–20. 8. Pereira RF, Halford KW, O’Hara MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci U S A. 1995;92:4857–4861. 9. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med. 2000;6:1282–1286.

10. Ralf H. Isolation of primary and immortalized CD34⫺ hematopoietic and mesenchymal stem cells from various sources. Stem Cells. 2000; 18:1–9. 11. Sandmaier BM, Storb R, Kniley J, et al. Evidence of allogeneic stromal engraftment in bone marrow using canine mesenchymal stem cells. Blood. 1998;92:116a (abst 473). 12. Devine S, Bartholomew A, Sturgeon C, et al. Studies of mesenchymal stem cells in non-human primates: evaluation of toxicity and engraftment. Blood. 1999;94:391a (abst 1733). 13. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol. 2000;18:307–316. 14. Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol. 1998;176:57–66. 15. Burgess AW, Wilson EM, Metcalf D. Stimulation by human placental conditional medium of hematopoietic colony formation by human marrow cells. Blood. 1977;49:573–583. 16. Kadereit S, Deeds LA, Haynesworth SE, et al. Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34⫹/ CD38– early progenitors cultured over human MSCs as a feeder layer. Stem Cells. 2002;20:573–582. 17. Steinborn A, Gall CV, Hildenbrand R, et al. Identification of placental cytokine-producing cells in term and preterm labor. Obstet Gynecol. 1998;91:329–35. 18. Melchers F. Murine embryonic B lymphocyte development in the placenta. Nature. 1979;277:219–221. 19. de Bruijin M, Speck NA, Peeters MC, Dzierzak E. Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo. EMBO J. 2000;19:2465–2474. 20. North T, de Bruijin MFTR, Stacy T, et al. Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo. Immunity. 2002;16:661–672. 21. Tavian M, Hallais MF, Peault B. Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo. Development. 1999; 126:793–803. 22. Mayani H, Lansdorp PM. Biology of human umbilical cord bloodderived hematopoietic stem/progenitor cells. Stem Cells. 1998;16: 153–165. 23. Alvarez-Silva M, Belo-Diabangouaya P, Salaun J, Dieterlen-Lievre F. Mouse placenta is a major hematopoietic organ. Development. 2003; 130:5437–5444. 24. Klyushnenkova EN, Mosca J, McIntosh KR. Human mesenchymal stem cells suppress allogeneic T cell responses in vitro: Implications for allogeneic transplantation. Blood. 1998;92:642a (abst 2652). 25. Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 2002;30:42–48. 26. DiNicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99:3838–3843. 27. Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood. 2003;101:3722–3729. 28. Tse WT, Beyer W, Pendleton JD, D’Andrea A, Guinan EC. Bone marrow derived mesenchymal stem cells suppress T cell activation without inducing allogeneic anergy. Blood. 2000;96:241a (abst 1034). 29. Lazarus HM, Haynesworth SE, Gerson SL, Rosenthal NS, Caplan AI. Ex vivo expansion and subsequent infusion of human bone marrowderived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant. 1995;16:557– 564. 30. Lazarus H, Curtin P, Devine S, et al. Role of mesenchymal stem cells (msc) in allogeneic transplantation: early phase I clinical results. Blood. 2000;96:392a (abst 1691).