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 detected by Alcian blue staining.Mixed lymphocyte reactions (MLR)MPCs and stimulators (peripheral blood