z' {2 }! _! u. b. r$ r$ WCorrespondence:Yumi Fukuchi, Ph.D., Division of Cellular Therapy, The Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Telephone: 81-3-5449-5759; Fax: 81-3-5449-5453; e-mail: yfukuchi@ims.u-tokyo.ac.jp3 w6 E% r1 p! m9 D, m$ k/ l
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ABSTRACT2 P, A& _/ H, ~( R. z6 l
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Multipotential mesenchymal stem/progenitor cells (MSCs) can be induced to differentiate into bone, adipose, cartilage, muscle, and endothelium if these cells are cultured under specific permissive conditions . In rodents, a specific type of MSC (termed multipotent adult progenitor cell) can be isolated from bone marrow (BM) and contributes to most somatic cell types when injected into early blastocysts at the single-cell level . Because MSCs have unique immunologic characteristics that suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo , persistence in a xenogeneic environment is favored . With such multiple differentiation capacities and unique immunoregulatory features plus self-renew potential , MSCs show promise as a possible therapeutic agent. Data from preclinical transplantation studies suggested that MSC infusions not only prevent the occurrence of graft failure but also have immunomodulatory effects . t$ m4 L) x/ I/ [ + H4 j% F, @$ s3 [MSCs are a rare population (approximately 0.001%–0.01%) of adult human BM . Moreover, numbers of BM MSCs significantly decrease with age . MSCs are also relatively few in adult peripheral blood and in term cord blood . A recent study showed that the population of MSC-like cells exists within the umbilical vein endothelial/ subendothelial layer . Furthermore, MSCs are present in fetal organs, such as liver, BM, and kidney, and circulate in the blood of preterm fetuses . However, fetal samples can be difficult to procure, and term cord blood compared with preterm is a poor source of MSCs . Such being the case, searching for appropriate sources, avoiding ethical issues, and establishing suitable culture systems are a challenge.9 V' N" Y$ M: g3 r3 J2 R1 x& V. V
% b, z+ P K6 u) Q& n- wIn this study, we evaluated the possibility that MSCs or cells with MSC-like potency are present in the human term placenta, and we obtained evidence that cells with the phenotype of MSCs exist in this tissue. / P6 L4 s% j, i1 e* ^4 |. L & ]% d2 c2 `" \1 c4 {" ^3 JMATERIALS AND METHODS7 Z" A4 I" N3 f2 Z
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Characterization of Placenta-Derived Cells 3 J3 |( a. s( {6 g* D& r# j; J# z! M7 s
Searching for alternative sources of MSCs, we attempted to prepare human term placentas and isolated fibroblast-like cells from every placenta isolation (n = 57; Fig. 1). In a single-cell suspension culture of the isolated placenta, cells firstly formed colony-forming unit fibroblast (CFU-F)–like colonies (Figs. 1A, b). On the other hand, in the culture of small trypsin-digested residues of placenta, cells began to migrate and proliferate (data not shown). After the first passage, cells from both samples expanded in the same monolayer manner (Fig. 1A, a–c). Cord blood (CB) is a rich source of hematopoietic stem cells and MSCs, and the term placenta contains much CB, primarily adherent cells derived from freshly isolated CB mononuclear cells (n = 77). However, CBs were obtained (after receiving the informed consent from Kiyosenomori Hospital, Tokyo) but did not survive in -MEM containing 15% FBS. To determine whether these cells were from maternal or fetal parts of the placenta, we did a fluorescence in situ hybridization analysis using X-and Y-probes. These cells were positive for X- and Y-signals, indicating that they were from a fetal part of the placenta (Fig. 1B). The placenta-derived cells were classified into two groups according to growth characteristics; one could proliferate more than 20 passages (Fig. 1C, Nos. 40 and 29), and the other went into replicative senescence between 10 and 20 passages (Fig. 1C, Nos. 41 and 44). The former type had a small and homogeneous morphology, but the latter type was of a bigger shape than the former. We also examined the surface marker profile of the above three representative placenta-derived cell lines using FACS, and these three lines had a similar phenotype, as follows: CD45lowCD31–AC133–CD54 CD29 CD44 (Fig. 1D), which closely resembles the phenotypes of BM-derived and CB-derived MSCs .& s9 B# q; f5 V8 P
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Figure 1. Isolation and characterization of placenta-derived cells. (A): Morphology of placenta-derived cells. Cells from a single-cell suspension easily expanded through the formation of colony-forming unit fibroblast–like colonies. a: 10 days after isolation (x100 magnification); b: 3 weeks after isolation (x40 magnification); c: 6 weeks after isolation (passage 3; x40 magnification). (B): Fluorescence in situ hybridization analysis for human X/Y chromosomes. Cells from male placenta have Y-positive (green) and X-positive (orange) signals. (C): Growth curve of placenta-derived cells. Frozen and thawed cells (n = 4, started at passage six or seven) were seeded at 0.5 x 105 cells per well and cultured until 90% confluence was reached. These cells were resuspended, enumerated, and reseeded at the same density for 10 days. (D): Immunophenotype of placenta-derived cells. Cells were stained with phycoerythrin-conjugated or fluorescein isothiocyanate–conjugated antibodies against CD45, CD31,AC133, CD54, CD29, CD44, or immunoglobulin isotype control antibodies. Cells were analyzed using fluorescence-activated cell sorter Calibur. Individual placenta-derived cells were given serial numbers of placenta isolation. Representative samples were used for these figures.) ^1 b9 d6 E+ t" O+ J
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Gene Expression Patterns of Placenta-Derived Cells6 D' C2 F& K+ ]; Z- P. Y% r, S
5 T1 Z) `7 c' D( [For a closer study of placenta-derived cells, we did a RT-PCR analysis for various genes, including stem cell markers, hematopoietic/endothelial cell–related genes, and organ-specific genes. The placenta-derived cells expressed many of the genes derived from mesoderm, ectoderm, and endoderm (Fig. 2). Additionally, expression patterns of stem cell markers and hematopoietic/endothelial cell–related genes in placenta-derived cells were similar to those of human BM (hBM)–derived MSCs (Fig. 2, lane 2). v5 K6 G0 t- M( b- B* ?/ p0 R. H: o0 |7 `( M# F0 x0 t
Figure 2. Gene expression patterns of placenta-derived cells. Placenta-derived cells were characterized using reverse transcription–polymerase chain reaction. Samples are as follows: lane 1, noncultured placenta (trypsin-digested residue); lane 2, human bone marrow–derived mesenchymal stem/progenitor cells; lanes 3–7, placenta (Nos. 29, 41, 42, 44, and 40)-derived cells; lane 8, reagent control; lane 9, positive control (i.e., Oct-4, Rex-1, HOXB4, and ?2-microglobulin were used for EoL-3. GATA-2, CBF?, TAL-1, CD34,AC133, flk-1, and flt-1 were used for TF-1. NKx2.5 and GATA-4 were used for human heart RNA. Renin and myogenin were used for human kidney RNA and skeletal muscle RNA, respectively. Nestin and GFAP were used for human brain RNA. -1-fetoprotein and albumin were used for human liver RNA. Amylase and insulin were used for human pancreas RNA). In this figure, we took up the data from the five representative placenta-derived cells, and each of placenta-derived cells was shown by serial numbers of placenta isolation., E0 y3 y( j q1 U; W( ?
) O1 z3 v4 B( S0 T7 l7 dDifferentiation Potential of Placenta-Derived Cells% q/ A1 J F! u% Z$ h
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To estimate the potential to differentiate into osteoblasts and adipocytes, the placenta-derived cells were cultured in osteogenic or adipogenic medium. At the end of the induction periods, most of the cells were Alizarin Red S–positive (Figs. 3B, 3C) or Oil Red O–positive (Figs. 3E, 3F), indicating differentiation to osteoblasts or adipocytes, respectively. In contrast, cells cultured with regular medium were not significantly stained (Figs. 3A, 3D). Such data indicate that the placenta-derived cells had bidirectional differentiation potency. ' O1 y% W: h$ Z. U! `8 V 2 R; ]) c, O" _Figure 3. Differentiation potential of placenta-derived cells. After a 2-week culture in osteogenic (B, C) or adipogenic (E, F) medium or regular medium (A, D), each of the placenta-derived cells was evaluated for osteogenic or adipogenic differentiation using specific staining and hematoxylin counterstaining. Magnification:A, B, D, E, x 40; C, F, x 100. A representative sample was used for this figure. , V3 p+ P3 M: ? w; i7 F$ I( M; s" c! Z3 s* U6 O
Subcloning of Placenta-Derived Cells $ J4 _' _; v& f9 q2 D) A& O; T6 \+ r1 k, N
The placenta-derived cells used in the above experiments are obviously heterogeneous and may be a mixture of progenitors that can differentiate into specific lineages. To exclude this possibility, we attempted to subclone No. 40 placenta–derived cells showing the human MSC (hMSC)–like gene expression pattern using RT-PCR (Fig. 2, lanes 2 and 7). We established two clones, B2 and F4 (Fig. 4A, lanes 1 and 9), which retained almost all of the phenotypes of their parental cells; surface marker expression (CD45lowCD31–AC133–CD54 CD29 CD44 ), gene expression patterns, and differentiation potential (Figs. 4B–4D versus Figs. 1–3). Moreover, these phenotypes were similar to those of other placenta-derived cell lines. Such data suggest that although the placenta-derived cells are considered to be polyclonal, most of the clones are similar in gene-expression profiles and retain the differentiation capacity to osteoblasts and adipocytes. 2 a# f& H; w1 p6 y! t$ D8 A9 L) e) L _1 x0 [0 d% [
Figure 4. Establishment and characterization of two clones from No. 40 placenta-derived cells. (A): Establishment of placenta-derived clones. No. 40 placenta-derived cells were transduced with MSCV-IRES-GFP retrovirus, and green fluorescent protein (GFP)-positive population was sorted by fluorescence-activated cell sorting, then replated onto a 96-well dish at 5 or 10 cells per well and expanded. DNAs from these GFP-positive No. 40 placenta-derived subclones were digested overnight with BamHI (cut only once in the MSCV-IRES-GFP plasmid), and fragments were separated by electrophoresis and probed with a 32P-labeled GFP cDNA probe. Samples are as follows for lanes 1-6, 8, and 9: subclones B2, B4, D2, D3, E4, G3, F1, and F4, respectively. These subclones were obtained from sub-cloning of five cells per ell. Lanes 7 and 10, subclones E4 and G4. These were obtained from subcloning of 10 cells per well. Subclones B2 (lane 1) and F4 (lane 9) had a single retroviral insert. (B): Immunophenotype of clones. Clones F4 and B2 were stained with phycoerythrin-conjugated antibodies against CD45, CD31,AC133, CD54, CD29, and CD44 or immunoglobulin isotype control antibodies then analyzed by fluorescence-activated cell sorter Calibur. (C): Gene expression patterns of clones. Clones F4 and B2 were characterized by reverse transcription–polymerase chain reaction analysis. Samples are as follows: lane 1, clone F4; lane 2, clone B2; lane 3, positive control (same positive controls were used in Fig. 2). (D): Differentiation potential of clones. Clones F4 and B2 were cultured in osteogenic (b, c, e, f), adipogenic (h, i, k, l), or regular medium (a, d, g, j) for 2 weeks. After the culture periods, each of the clones was evaluated for osteogenic or adipogenic differentiation using specific staining and hematoxylin counterstaining. Magnification: a, b, d, e, g, h, j, k, x 40; c, f, i, l, x 100. 6 D- [8 e. Y$ | 5 d0 d) ^/ Z7 \% B, kDISCUSSION+ G0 o4 D' h2 G( R# K ?
( h- [* b+ _) z% A& d- AWe thank Drs. T. A. Takahashi and N. Watanabe (Division of Cell Processing, Institute of Medical Science, The University of Tokyo, Japan) for significant advice on these placenta-derived cells; Dr. Y. Koshino and Ms. M. Ito (Division of Cellular Therapy, Institute of Medical Science, The University of Tokyo, Japan) for advice and technical support; and Drs. H. Funabiki and S. Akutsu (Kiyosenomori Hospital, Japan) for assistance with cord blood collections. 7 O# \' b- X# } / j9 [9 n9 t# r; {3 s' vREFERENCES 3 o) h" B8 `; o) H4 [0 k $ [8 M) C( V6 m" ALiechty 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. U. S j# @3 ?/ F
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Clark E, Wognum AW, Marciniak R et al. Mesenchymal cell precursors from human bone marrow have a phenotype that is direct from cultured mesenchymal cells and are exclusively present in a small subset of CD45lo SH2 cells. Blood 2001;98:85a.(Yumi Fukuchi, Hideaki Nak)作者: lqzou918 时间: 2009-8-24 15:44