单个细胞来源胎盘间充质干细胞培养方法的比较研究
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摘要
目的·观察胚胎来源间充质干细胞(placental derived mesenchymal stem cells,PMSCs)在人血小板裂解液(human platelet lysate,HPL)、碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF)及传统的胎牛血清(fetal bovine serum,FBS)不同组合的培养基中的倍增数目及细胞性状,着重探索更合适的PMSCs培养体系。方法·分离单个细胞来源PMSCs,扩增后分别用 4 种培养基孵育,分为FBS 组、FBS+b FGF组、HPL组以及HPL+b FGF组。观察细胞形态及生长状态、细胞表型及多能分化。在P1、P2、P3、P4代进行计数。取P4代细胞接种,分析集落形成单位的个数。结果·经鉴定,PMSCs具有间充质干细胞的生物特性。数量上,FBS+b FGF组在P4代达到(1.12×10~7)个/cm~2,HPL组达到(1.24×10~7)个/cm~2(P>0.05),而FBS组和HPL+b FGF组则只有(5.58×10~6)个/cm~2和(8.56×10~6)个/cm~2。细胞形态上,FBS+b FGF组与HPL组 P4代的 PMSCs均保持贴壁生长,但HPL组细胞于P5代无法贴壁生长。细胞集落数量上,P4代时,FBS+b FGF组为51个/ 孔,HPL为52个/ 孔(P>0.05)。细胞生物学特性上,FBS+b FGF 组和HPL组在P4代时均能基本保有间充质干细胞的特性和多能分化的能力。结论·从细胞数量上来看,HPL组中的P4代细胞数与目前常用的FBS+b FGF培养基没有显著差异,且已达到临床治疗剂量需求;从细胞生物学特性上来看,HPL培养基与FBS+b FGF培养基均能基本保持PMSCs细胞特性和多能分化的能力。但由于HPL培养基免疫原性低,因此更适合临床移植应用。然而,由于HPL培养基无法维持PMSCs生长至5代以后,故无法用于大规模细胞扩增。HPL+b FGF在目前实验条件下并不具备优势。
Objective To explore optimal placental derived mesenchymal stem cells (PMSCs) culture medium using different combination of human platelet lysate (HPL), basic fibroblast growth factor (b FGF) and traditional fetal bovine serum (FBS) for further basic and clinical study. Methods Single cell derived PMSCs was harvested and incubated with 4 kinds of culture, i.e, FBS, FBS+b FGF, HPL and HPL+b FGF. The morphology, growth state, cell phenotype and multi-energy differentiation were observed. Cells of P1, P2, P3 and P4 generation were counted respectively. The number of units of colony formation was analyzed by inoculating P4 cells. Results It was identified that PMSCs had the biological properties of MSCs. Quantificationally, cell density reached (1.12×10~7) cells/cm~2 in FBS+b FGF group and (1.24×10~7) cells /cm~2 in HPL group (P>0.05) in P4, while those in FBS group and HPL+b FGF group were (5.58×10~6) cells /cm~2 and (8.56×10~6) cells /cm~2, respectively. For cell morphology, the P4 PMSCs of FBS+b FGF and HPL groups kept adherent growth, but the HPL cells could not be adherent in P5 generation. The number of colony was 51/well in FBS+b FGF group, and it was 52/well in HPL group (P>0.05) in P4. The FBS+b FGF group and the HPL group were able to maintain the characteristics of MSCs and the ability of pluripotent differentiation in the P4 generation. Conclusion PMSCs in P4 cultured in HPL medium can keep the biological characteristics and meet the clinical transplantation requirements in quality and quantity, which are preferable for their low immunogenicity to clinical applications. In long term, PMSCs cultured in FBS+b FGF medium are preferable for the longer lasting characters of MSCs and larger quantity in basic studies. HPL+b FGF medium has no advantage on quality and quantity.
Objective To explore optimal placental derived mesenchymal stem cells (PMSCs) culture medium using different combination of human platelet lysate (HPL), basic fibroblast growth factor (b FGF) and traditional fetal bovine serum (FBS) for further basic and clinical study. Methods Single cell derived PMSCs was harvested and incubated with 4 kinds of culture, i.e, FBS, FBS+b FGF, HPL and HPL+b FGF. The morphology, growth state, cell phenotype and multi-energy differentiation were observed. Cells of P1, P2, P3 and P4 generation were counted respectively. The number of units of colony formation was analyzed by inoculating P4 cells. Results It was identified that PMSCs had the biological properties of MSCs. Quantificationally, cell density reached (1.12×10~7) cells/cm~2 in FBS+b FGF group and (1.24×10~7) cells /cm~2 in HPL group (P>0.05) in P4, while those in FBS group and HPL+b FGF group were (5.58×10~6) cells /cm~2 and (8.56×10~6) cells /cm~2, respectively. For cell morphology, the P4 PMSCs of FBS+b FGF and HPL groups kept adherent growth, but the HPL cells could not be adherent in P5 generation. The number of colony was 51/well in FBS+b FGF group, and it was 52/well in HPL group (P>0.05) in P4. The FBS+b FGF group and the HPL group were able to maintain the characteristics of MSCs and the ability of pluripotent differentiation in the P4 generation. Conclusion PMSCs in P4 cultured in HPL medium can keep the biological characteristics and meet the clinical transplantation requirements in quality and quantity, which are preferable for their low immunogenicity to clinical applications. In long term, PMSCs cultured in FBS+b FGF medium are preferable for the longer lasting characters of MSCs and larger quantity in basic studies. HPL+b FGF medium has no advantage on quality and quantity.
引文
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[22]LüFJ,Tuan RS,Cheung KM,et al.Concise review:the surface markers and identity of human mesenchymal stem cells[J].Stem Cells,2014,32(6):1408-1419.
[2]Jeon YJ,Kim J,Cho J,et al.Comparative analysis of human mesenchymal stem cells derived from bone marrow,placenta,and adipose tissue as sources of cell therapy[J].J Cell Biochem,2016,117(5):1112-1125.
[3]Luan X,Li G,Wang G,et al.Human placenta-derived mesenchymal stem cells suppress T cell proliferation and support the culture expansion of cord blood CD34+cells:a comparison with human bone marrow-derived mesenchymal stem cells[J].Tissue Cell,2013,45(1):32-38.
[4]Zhao YM,Li JY,Lan JP,et al.Cell cycle dependent telomere regulation by telomerase in human bone marrow mesenchymal stem cells[J].Biochem Biophys Res Commun,2008,369(4):1114-1119.
[5]Wei LL,Gao K,Liu PQ,et al.Mesenchymal stem cells from Chinese Guizhou minipig by h TERT gene transfection[J].Transplant Proc,2008,40(2):547-550.
[6]Simonsen JL,Rosada C,Serakinci N,et al.Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells[J].Nat Biotechnol,2002,20(6):592-596.
[7]白金萍,李秀英,李雪,等.胎盘间充质干细胞传代后的增殖能力[J].中国组织工程研究,2014,18(10):1591-1596.
[8]Schmidt D,Joyce EJ,Kao WJ.Fetal bovine serum xenoproteins modulate human monocyte adhesion and protein release on biomaterials in vitro[J].Acta Biomater,2011,7(2):515-525.
[9]Eastment CT,Sirbasku DA.Human platelet lysate contains growth factor activities for established cell lines derived from various tissues of several species[J].In Vitro,1980,16(8):694-705.
[10]Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement[J].Cytotherapy,2006,8(4):315-317.
[11]Blande IS,Bassaneze V,Lavini-Ramos C,et al.Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate[J].Transfusion,2009,49(12):2680-2685.
[12]Crespo-Diaz R,Behfar A,Butler GW,et al.Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability[J].Cell Transplant,2011,20(6):797-811.
[13]Abdelrazik H,Spaggiari GM,Chiossone L,et al.Mesenchymal stem cells expanded in human platelet lysate display a decreased inhibitory capacity on T-and NK-cell proliferation and function[J].Eur J Immunol,2011,41(11):3281-3290.
[14]Salzig D,Leber J,Merkewitz K,et al.Attachment,growth,and detachment of human mesenchymal stem cells in a chemically defined medium[J].Stem Cells Int,2016,2016:5246584.
[15]Mehrzadi S,Safa M,Kamrava SK,et al.Protective mechanisms of melatonin against hydrogen peroxide induced toxicity in human bone-marrow derived mesenchymal stem cells[M].Can J Physiol Pharmacol,2017,95(7):773-786.,
[16]Luchetti F,Canonico B,Bartolini D,et al.Melatonin regulates mesenchymal stem cell differentiation:a review[J].J Pineal Res,2014,56(4):382-397.
[17]Ercan E,Bagla AG,Aksoy A,et al.In vitro protection of adipose tissue-derived mesenchymal stem cells by erythropoietin[J].Acta Histochem,2014,116(1):117-125.
[18]Ye L,Chen L,Yu Q,et al.Effect of recombinant human erythropoietin on the stemness of bone marrow-derived mesenchymal stem cells in vitro[J].Int J Stem Cells,2010,3(2):175-182.
[19]Bischoff DS,Makhijani NS,Yamaguchi DT,et al.Constitutive expression of human telomerase enhances the proliferation potential of human mesenchymal stem cells[J].Bio Res Open Access,2012,1(6):273-279.
[20]Madonna R,Taylor DA,Geng YJ,et al.Transplantation of mesenchymal cells rejuvenated by the overexpression of telomerase and myocardin promotes revascularization and tissue repair in a murine model of hindlimb ischemia[J].Circ Res,2013,113(7):902-914.
[21]Okada M,Kim HW,Matsu-ura K,et al.Abrogation of age-induced micro RNA-195 rejuvenates the senescent mesenchymal stem cells by reactivating telomerase[J].Stem Cells,2016,34(1):148-159.
[22]LüFJ,Tuan RS,Cheung KM,et al.Concise review:the surface markers and identity of human mesenchymal stem cells[J].Stem Cells,2014,32(6):1408-1419.