胎儿心脏黏附细胞具有类似间充质祖细胞特征

1、胎儿心脏黏附细胞具有类似间充质祖细胞特征             作者：江小霞，苏永锋，李秀森，张毅，吴英，毛宁【摘要】  为了观察人心脏是否含具有间充质祖细胞特性的细胞，从胎儿心脏分离、培养单个核细胞并从形态、表型和功能3个方面与骨髓间充质祖细胞进行比较和鉴定。结果表明，从心脏分离培养的细胞为成纤维样，表面抗原为CD73, CD105, CD29, CD44, HLA-ABC, CD166 阳性，而CD45, CD34, CD86, HLA-DR 阴性。在不同的分

2、化体系中，细胞能分化为脂肪细胞、成骨细胞和软骨细胞。细胞扩增迅速，具有低免疫原性特性。结论：从心脏分离培养的细胞具有间充质祖细胞特性。 【关键词】  胎儿心脏； 心脏黏附细胞； 间充质祖细胞Human Fetal Heart-derived Adherent Cells with Characteristics Similar to Mesenchymal Progenitor CellsAbstractThis study was aimed to  investigate if human heart harbored a population of primitive

3、 undifferentiated cells with the characteristics of  MPC. Cells were isolated from human fetal heart and were cultured under conditions appropriate for bone marrow-derived MPCs. Their morphology, phenotypes and functions were tested by methods developed for MPC from other sources. The results s

4、howed that morphologically, cells were spindle shaped and resembled fibroblasts. In their undifferentiated state, cells were CD73, CD105, CD29, CD44, HLA-ABC, CD166 positive and CD45, CD34, CD86, HLA-DR negative. When cultured in adipogenic, osteogenic or chondrogenic media, cells differentiated int

5、o adipocytes, osteocytes and chondrocytes respectively. They could be extensively expanded in vitro and exhibited very low immunogenicity as evaluated by T cell proliferation assays. It is concluded that  cells isolated from fetal heart possess simi-larity to their adult and fetal bone marrow c

6、ounterparts in morphologic, immunophenotypic, and functional characteristics.Key wordsfetal heart; heart derived adherent cell; mesenchymal progenitor cellBone marrow-derived mesenchymal progenitor cells (MPC) have attracted great attention because of their capability for renewal and differentiation

7、 into various lineages of mesenchymal tissues1 and their potential platforms for the systemic delivery of therapeutic proteins in vivo following gene transfer using oncogenic retroviruses2. Studies involving a variety of animal models have shown that adult bone marrow-derived MPC can migrate and eng

8、raft in numerous organs and differentiate along tissue-specific lineages under the stimulation of local factors, and may be useful in the repair or regeneration of damaged or mutated bone, cartilage, or myocardial tissues3-5.Recent work has shown that MPC are present in many tissues, including umbil

9、ical cord6, umbilical cord blood7, bone marrow, fetal blood, and fetal li-ver8. Though the use of MPC in the treatment of acute myocardial infarction has become a novel therapeutic option, the knowledge of their presence in heart is limited 9. Our aim was to investigate whether there were cells with

10、 the characteristics of MPC in human fetal heart.Materials and MethodsIsolation and culture of adult and fetal bone mar-row, fetal heart mesenchymal progenitor cellsThe Research Ethics Committees of Xuanwu Hospital approved human tissue for research purposes.Human fetal heart samples were obtai

11、ned from accidental abortus of 4.0-5.0 months under consent. Single-cell suspensions of fetal heart mesenchymal tissue were prepared by carefully rinsing out of the blood and mincing myocardial tissue, away from the blood vessel, through a 70-m-nylon filter. Then cells from adult and fetal bone marr

12、ow samples  and fetal heart samples were diluted  by using  10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) in Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG; Gibco BRL, Life Technologies, Paisley, United Kongdom) with 50 U/ml penicillin, 50 g/ml streptomycin, a

13、nd 2 mmol/L L-glutamine. Cells were plated into 6-well plate at a density of 100 000 cells/cm2 and incubated at 37  in 5% CO2.  After 36 hours, nonadherent cells were removed, and the medium was replaced. At 80% confluence, cells were harvested with 0.25% trypsin and 2 mmol/L EDTA for 5 mi

14、nutes at 37 and were replated in 75-cm2 flasks. To expand the cells through successive passages, they were plated at 104 cells/cm2, grown to near confluence, and harvested with the same protocol.Fluorescence-activated cell sorting (FACS) analysis of cultured cellsMonolayer adherent cells from adult

15、bone marrow (n = 4), fetal bone marrow (n = 4), and fetal heart (n = 4) were trypsinized and labbled with anti-CD73-phycoerythrin (PE; PharMingen, USA), CD105-fluorescein isothiocyanate (FITC; Serotec, Oxford, United Kingdom), HLA-ABC-FITC, CD44-PE, CD29-PE, CD166-PE, CD45-FITC, CD34-PE, HLA-DR-FITC

16、, CD86-PE (Becton Dickinson, USA) and were analyzed by flow cytometry (Beckman Coulter, USA).Adipogenic, osteogenic, and chondrogenic diffe- rentiationAdipogenic differentiation was assessed by incubation with DMEM with 10% FBS supplemented with 1 mol/L dexamethasone, 10 g/ml insulin, 0.5 mmol/L iso

17、butyl methylxanthine, and 200 mol/L indomethacin (Sigma, St. Louis, MO, USA) for 2 weeks. The presence of adipocytes was assessed by the cellular accumulation of neutral lipid vacuoles that stained with Oil red O (Sigma). Osteogenic differentiation was assessed by culturing cells in an osteogenic me

18、dium (DMEM with 10% FBS supplemented with 0.1 mol/L dexamethasone, 50 mol/L ascorbic acid, and 10 mmol/L -glycerol phosphate). The onset of osteoblasts was evaluated by calcium accumulation (von Kossa staining). Medium with DMEM containing 2.5% FBS, 50 ng/mL transforming growth factor-1 (Peprotech,

19、Rocky Hill, NJ, USA), 50 g/ml ascorbic acid, 1 mmol/L sodium pyruvate, 6.25 g/ml bovine insulin, 6.25 g/ml transferrin, 6.25 g/ml selenious acid, and 1.25 g/ml bovine serum albumin was used for chondrogenic differentiation. Extracellular matrix used to assess chondrogenic differentiation, was detect

20、ed by Alcian blue staining.Mixed lymphocyte reactions (MLR)MPCs and stimulators (peripheral blood mononuclear cells, PBMNC) were irradiated (30 Gy) before being cultured with T lymphocytes. CD3+ T cells purified from PBMNC by using the MACS CD3 isolation kit (Miltenyi Biotec) in  5×104/wel

21、l were mixed at diffe-rent ratio with MPCs in 96-well culture plates to ensure efficient cell-cell contact for 4 days in 0.2 ml RPMI 1640 medium (Gibco BRL) containing 20% heat-inactivated FBS. T-cell proliferation was measured on day 3 by means of an 16-hour pulse with 3H-thymidine(3H-TdR)  1&

22、#160; Ci/well. 3H-TdR incorporation was measured by using a liquid scintillation counter. The experiments were performed for at least 3 times.百事通 ResultsMorphology and immunophenotype characteristics of cells from human fetal heartNucleated cells from human fetal heart plated at low-density formed i

23、ndividual colonies displaying fibroblast-like morphology. After subcultivation,  nucleated cells proliferated with a population-doubling time of 23 hours and reached a confluent condition. Adherent cells could be readily expanded in vitro by means of successive cycles of trypsinization, seeding

24、. And culture every 3 days for 20 passages displayed no visible changes in terms of their morphology under light microscopy. The immunophenotype of cells from adult and fetal bone marrow and fetal heart was determined by flow cytometry. The staining pattern of the cells was similar. As shown in Figu

25、re 1, adherent cells isolated from adult and fetal bone marrow and fetal heart were positive for CD73, CD105, HLA-ABC, CD44, CD29, CD166 and lacked of expression of CD45, CD34, CD86, HLA-DR. The phenotypic profile of adult, fetal bone marrow and fetal heart adherent cells did not change after 12 pas

26、sages in culture.Differentiation ability of cells from human fetal heart Adipogenic differentiation was apparent after 1 week of incubation; two weeks later  intracellular lipid accumulation was visualized using Oil Red O staining (Figure 2. A, B, C). Deposition of mineralized matrix on the cul

27、ture vessels was shown by von Kossa staining (Figure 2. E, F, G). The staining results indicated the differentiation of cultured cells into osteocytic lineage. The positive alcian blue staining (Figure 2. I, J, K) suggested the expression of type II collagen of chondrocyte.Control cells did not show

28、 spontaneous adipocyte, osteoblast or chondrocyte formation even after 3-4 weeks of cultivation (Figure 2. D, H, L)Immunosuppressive effect of cells from human fetal heart in vitroThe MLR data  suggested a nonspecific immunosuppressive effect of cells from bone marrow and fetal heart  on&#

29、160; human CD3+ T cell proliferation in a dose dependent manner  (Figure 3). DiscussionIn this study we demonstrated that there were adherent cells with the characteristics of MPC resided in human fetal heart. They showed fibroblast like morphology and, like MPC from bone marrow, were posi

30、tive for some mesenchymal markers, such as CD73 (SH2, SH3), CD105 (SH4) and negative for the endothelial/hematopoietic progenitor marker CD34 and the pan leukocyte marker CD45. This meant that there were not endothelial progenitor cells (EPC), which expressed CD45 and CD34. In addition, the adherent

31、 cells had similar ability to differentiate into adipocyte, osteoblast and chondrocyte. Finally and most importantly, as MPC, the adherent cells exerted an immunosuppressive effect on T cells that was beneficial for clinical application. The out-dated view was that the heart lacked a pool of stem ce

32、lls capable of self-renewal and differentiation. But more and more evidences show that the adult heart, like the brain, is composed of mainly terminally differentiated parenchyma cells not reentering the cell cycle, is no doubt a terminally differentiated organ but containing adult stem cells suppor

33、ting its regeneration. Exciting new evidence has emerged that the transplanted human heart harbors a population of primitive undifferentiated cells derived from both the recipient and the donor. These primitive cells may be cardiac stem cells and may play a pivotal role in the remodeling process fol

34、lowing transplantation10. And recently, Beltrami AP et al11 have isolated cardiac stem cells from adult rat, which showed in vitro and in vivo self-renewing and multipotent. What is  more, in vivo manipulation of these stem cells could regenerate large amounts of functional myocardium, shown to

35、 be one of the most extensive solid organ tissue regenerations by using stem cells reported so far. Maybe autologous cardiac-specific stem cells are more beneficial to clinical cell therapy for cardiac diseases. The adherent cells we isolated are not the same as the cardiac stem cell. Though they bo

36、th are negative for CD34, CD45, cardiac stem cells do not express fibronectin and vimentin, which is different from the adherent cells (data no shown). The heart and the bone marrow, cardiomyocytes as well as bone marrow MPC, are of mesodermal origin, so we postulate that there will be MPC in adult

37、heart. But it needs further study to make things clearer.【参考文献】  1 Gerson SL. Mesenchymal stem cells: no longer second class marrow citizens. Nat Med, 1999; 5: 262-2642 Zhang XY, La-Russa VF, Bao L, et al. Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells. Mol Ther, 2002; 5(5 Pt 1): 555-5653 Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplanta