胶原支架促进人类多能干细胞向红细胞的诱导分化
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摘要
目的建立人胚胎干细胞(hESCs)来源的造血干/组细胞向红细胞分化的体外3D培养方法。方法hESCs与小鼠主动脉-性腺-中肾区(AGM-S3)基质细胞共培养14 d后获得CD34~+CD45~+造血干/祖细胞,对获得的CD34~+CD45~+细胞扩增5 d后,以相同初始接种数量分别接种于胶原水凝胶、透明质酸水凝胶、Ⅰ型胶原蛋白构建的三维(3D)支架材料(简称胶原支架)中,在体外模拟骨髓微环境,做红细胞定向分化,以普通液体悬浮培养(简称液体培养)为对照,通过MGG染色、流式细胞术、免疫染色、qRT-PCR等手段分别对收获的细胞做形态学、红系特异性表面标志表达情况、红系细胞成熟程度以及红系细胞发育过程中相关基因的表达情况分析。结果红系定向分化14 d后,胶原支架中收获的总细胞数量分别是液体培养、透明质酸水凝胶、胶原水凝胶的1.50、1.19、1.33倍,GPA~+CD71~+细胞数量分别是液体培养、透明质酸水凝胶、胶原水凝胶的1.55、1.25及1.48倍;GPA~+CD36~+细胞数量分别是液体培养、透明质酸水凝胶、胶原水凝胶的1.65、1.07、1.36倍。结论利用Ⅰ型胶原蛋白构建的3D支架材料可模拟骨髓微环境,促进红细胞分化。
Objective To establish anin vitrothree-dimensional(3D) microenvironment favoring the differentiationand proliferation of hematopoietic progenitors,derived from humanembryonicstemcells(hESCs),towards erythroblasts with biomimetic macroporous hydrogel scaffolds mimicking the bone marrow architecture. Methods According to the methods previously reported,we co-cultured hESCs with the mice aorta-gonad-mesonephros(AGM-S3) stromal cell,allowing for efficient acquisition of CD34~+ CD45~+ hematopoietic progenitors. Hyaluronic acid hydrogel,type Ⅰ collagen hydrogel and type Ⅰ collagen hydrogel scaffold with microporous structures were synthesized to replicate the in vivo physical environment and functioned as the housing frame for the culture of these progenitors withconventional liquid culture being the control group. Erythroblasts were collected from these culture models andanalyzed by May-Grünwald-Giemsa staining,flow cytometry analysis and immunochemical staining.The expression of hematopoiesis-relatedgeneswas observed by qRT-PCR.Results After 14 days of culture,the total cell number harvested from thecollagen scaffold model was 1. 50 times,1. 19 times and 1. 33 times ofthe cell numbers of the liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively.The cell number of GPA~+ CD71~+ cells harvested from thecollagen scaffold model was 1. 55 times,1. 25 times and 1. 48 times of the cell numbersthe liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively.The cell number of GPA~+ CD36~+ cells harvested from the collagen scaffold model was 1. 65 times,1. 07 times and 1. 36 times of the cell numbers from the liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively. Conclusion we have successfully developed a microporous-scaffold culture modelthat partially mimicked the bone marrow microenvironment in vivo. This model can promote the proliferation of erythroblasts in vitro.
Objective To establish anin vitrothree-dimensional(3D) microenvironment favoring the differentiationand proliferation of hematopoietic progenitors,derived from humanembryonicstemcells(hESCs),towards erythroblasts with biomimetic macroporous hydrogel scaffolds mimicking the bone marrow architecture. Methods According to the methods previously reported,we co-cultured hESCs with the mice aorta-gonad-mesonephros(AGM-S3) stromal cell,allowing for efficient acquisition of CD34~+ CD45~+ hematopoietic progenitors. Hyaluronic acid hydrogel,type Ⅰ collagen hydrogel and type Ⅰ collagen hydrogel scaffold with microporous structures were synthesized to replicate the in vivo physical environment and functioned as the housing frame for the culture of these progenitors withconventional liquid culture being the control group. Erythroblasts were collected from these culture models andanalyzed by May-Grünwald-Giemsa staining,flow cytometry analysis and immunochemical staining.The expression of hematopoiesis-relatedgeneswas observed by qRT-PCR.Results After 14 days of culture,the total cell number harvested from thecollagen scaffold model was 1. 50 times,1. 19 times and 1. 33 times ofthe cell numbers of the liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively.The cell number of GPA~+ CD71~+ cells harvested from thecollagen scaffold model was 1. 55 times,1. 25 times and 1. 48 times of the cell numbersthe liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively.The cell number of GPA~+ CD36~+ cells harvested from the collagen scaffold model was 1. 65 times,1. 07 times and 1. 36 times of the cell numbers from the liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively. Conclusion we have successfully developed a microporous-scaffold culture modelthat partially mimicked the bone marrow microenvironment in vivo. This model can promote the proliferation of erythroblasts in vitro.
引文
[1]Chang KH,Nelson AM,Cao H,et al,Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin,Blood,2006,108(5):1515-1523.
[2]Ma F,Ebihara Y,Umeda K,et al,Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis,Proc Natl Acad Sci USA,2008,105(35):13087-13092.
[3]Lu SJ,Feng Q,Park JS,et al.Biologic properties and enucleation of red blood cells from human embryonic stem cells,Blood,2008,112(12):4475-4484.
[4]Chow A,Huggins M,Ahmed J,et al:CD169+macrophages provide a niche promoting erythropoiesis under homeostasis and stress,Nat Med,2013,19(4):429-436.
[5]Scadden DT.The stem cell niche in health and leukemic disease,Best Pract Res Clin Haematol,2007,20(1):19-27.
[6]Ferreira MS,Jahnen-Dechent W,Labude N,et al.Cord bloodhematopoietic stem cell expansion in 3D fibrin scaffolds with stromal support,Biomaterials,2012,33(29):6987-6997.
[7]Mortera-Blanco T,Mantalaris A,Bismarck A,et al.Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds,Biomaterials,2011,32(35):9263-9270.
[8]Choi JS,Harley BAC.The combined influence of substrate elasticity and ligand density on the viability and biophysical properties of hematopoietic stem and progenitor cells,Biomaterials,2012,33(18):4460-4468.
[9]Feng Q,Chai C,Jiang XS,et al.Expansion of engrafting human hematopoietic stem/progenitor cells in three-dimensional scaffolds with surface-immobilized fibronectin,J Biomed Mater Res A,2006,78(4):781-791.
[10]Lutolf MP,Gilbert PM,Blau HM.Designing materials to direct stem-cell fate.Nature,2009,462(7272):433-441.
[11]Ueda T,Fujita A,Ogawa R,et al.Adipose-derived stromal cells grown on a hydroxyapatite scaffold can support hematopoiesis in regenerated bone marrow in vivo,Cell Biol Int,2014,38(6):790-798.
[12]Fujita A,Migita M,Ueda T,et al.Hematopoiesis in regenerated bone marrow within hydroxyapatite scaffold,Pediatr Res,2010,68(1):35-40.
[13]Leisten I,Kramann R,Ventura FM,et al.3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche.Biomaterials,2012.33(6):p.1736-1747.
[14]Ribeil J,Zermati Y,Vandekerckhove J,et al.Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1,Nature,2007,445(7123):102-105.
[15]Lanneau D,de Thonel A,Maurel S,et al.Apoptosis versus cell differentiation:role of heat shock proteins HSP90,HSP70 and HSP27,Prion,2007,1(1):53-60.
[16]Telen MJ.Erythrocyte adhesion receptors:blood group antigens and related molecules,Transfus Medi Rev,2005,19(1):32-44.
[17]Xu H,Ito T,Tawada A,et al.Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72,J Biol Chem,2002,277(19):17308-17314.
[18]曾宪波,毛斌,赖默温,等.透明质酸水凝胶三维培养促进人胚胎干细胞向红系细胞诱导分化.中国输血杂志,2017,30(1):12-16.
[19]Mao B,Huang S,Lu X,et al:Early Development of Definitive Erythroblasts from Human Pluripotent Stem Cells Defined by Expression of Glycophorin A/CD235a,CD34,and CD36,Stem Cell Reports,2016,7(5):869-883.
[20]郁金凤,孙文翠,路旭琳,等.人胚胎干细胞高效诱导产生巨噬细胞方法的建立.中国输血杂志,2015,28(5):482-487.
[21]Housler GJ,Miki T,Schmelzer E,et al.Compartmental hollow fiber capillary membrane-based bioreactor technology for in vitro studies on red blood cell lineage direction of hematopoietic stem cells,Tissue Eng Pt C:Methods,2012,18(2):133-142.
[22]Timmins NE,Athanasas S,Nther MG,et al.Ultra-high-yield manufacture of red blood cells from hematopoietic stem cells,Tissue Eng Pt C:Methods,2011,17(11):1131-1137.
[23]Lee E,Han S Y,Choi HS,etal:Red blood cell generation by three-dimensional aggregate cultivation of late erythroblasts,Tissue Eng Pt A,2015,21(3-4):817-828.
[24]毛斌,马峰.h ESCs/hi PSCs体外诱导产生红细胞的研究进展及其临床应用的展望,中国细胞生物学学报,2012,34(11):1067-1079.
[25]Demange E,Kassim Y,Petit C,et al.Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry,J Tissue Eng Regen Med,2013,7(11):901-910.
[2]Ma F,Ebihara Y,Umeda K,et al,Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis,Proc Natl Acad Sci USA,2008,105(35):13087-13092.
[3]Lu SJ,Feng Q,Park JS,et al.Biologic properties and enucleation of red blood cells from human embryonic stem cells,Blood,2008,112(12):4475-4484.
[4]Chow A,Huggins M,Ahmed J,et al:CD169+macrophages provide a niche promoting erythropoiesis under homeostasis and stress,Nat Med,2013,19(4):429-436.
[5]Scadden DT.The stem cell niche in health and leukemic disease,Best Pract Res Clin Haematol,2007,20(1):19-27.
[6]Ferreira MS,Jahnen-Dechent W,Labude N,et al.Cord bloodhematopoietic stem cell expansion in 3D fibrin scaffolds with stromal support,Biomaterials,2012,33(29):6987-6997.
[7]Mortera-Blanco T,Mantalaris A,Bismarck A,et al.Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds,Biomaterials,2011,32(35):9263-9270.
[8]Choi JS,Harley BAC.The combined influence of substrate elasticity and ligand density on the viability and biophysical properties of hematopoietic stem and progenitor cells,Biomaterials,2012,33(18):4460-4468.
[9]Feng Q,Chai C,Jiang XS,et al.Expansion of engrafting human hematopoietic stem/progenitor cells in three-dimensional scaffolds with surface-immobilized fibronectin,J Biomed Mater Res A,2006,78(4):781-791.
[10]Lutolf MP,Gilbert PM,Blau HM.Designing materials to direct stem-cell fate.Nature,2009,462(7272):433-441.
[11]Ueda T,Fujita A,Ogawa R,et al.Adipose-derived stromal cells grown on a hydroxyapatite scaffold can support hematopoiesis in regenerated bone marrow in vivo,Cell Biol Int,2014,38(6):790-798.
[12]Fujita A,Migita M,Ueda T,et al.Hematopoiesis in regenerated bone marrow within hydroxyapatite scaffold,Pediatr Res,2010,68(1):35-40.
[13]Leisten I,Kramann R,Ventura FM,et al.3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche.Biomaterials,2012.33(6):p.1736-1747.
[14]Ribeil J,Zermati Y,Vandekerckhove J,et al.Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1,Nature,2007,445(7123):102-105.
[15]Lanneau D,de Thonel A,Maurel S,et al.Apoptosis versus cell differentiation:role of heat shock proteins HSP90,HSP70 and HSP27,Prion,2007,1(1):53-60.
[16]Telen MJ.Erythrocyte adhesion receptors:blood group antigens and related molecules,Transfus Medi Rev,2005,19(1):32-44.
[17]Xu H,Ito T,Tawada A,et al.Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72,J Biol Chem,2002,277(19):17308-17314.
[18]曾宪波,毛斌,赖默温,等.透明质酸水凝胶三维培养促进人胚胎干细胞向红系细胞诱导分化.中国输血杂志,2017,30(1):12-16.
[19]Mao B,Huang S,Lu X,et al:Early Development of Definitive Erythroblasts from Human Pluripotent Stem Cells Defined by Expression of Glycophorin A/CD235a,CD34,and CD36,Stem Cell Reports,2016,7(5):869-883.
[20]郁金凤,孙文翠,路旭琳,等.人胚胎干细胞高效诱导产生巨噬细胞方法的建立.中国输血杂志,2015,28(5):482-487.
[21]Housler GJ,Miki T,Schmelzer E,et al.Compartmental hollow fiber capillary membrane-based bioreactor technology for in vitro studies on red blood cell lineage direction of hematopoietic stem cells,Tissue Eng Pt C:Methods,2012,18(2):133-142.
[22]Timmins NE,Athanasas S,Nther MG,et al.Ultra-high-yield manufacture of red blood cells from hematopoietic stem cells,Tissue Eng Pt C:Methods,2011,17(11):1131-1137.
[23]Lee E,Han S Y,Choi HS,etal:Red blood cell generation by three-dimensional aggregate cultivation of late erythroblasts,Tissue Eng Pt A,2015,21(3-4):817-828.
[24]毛斌,马峰.h ESCs/hi PSCs体外诱导产生红细胞的研究进展及其临床应用的展望,中国细胞生物学学报,2012,34(11):1067-1079.
[25]Demange E,Kassim Y,Petit C,et al.Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry,J Tissue Eng Regen Med,2013,7(11):901-910.