Cellular and Molecular Biology of the Hepatic Stem Cell Compartment

肝干细胞室的细胞和分子生物学

• 批准号: 8552575
• 负责人: Snorri Thorgeirsson
• 金额: $ 12.72万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552575
• 关键词: Activation Analysis; Address; Adopted; Adult; Area; Bile fluid; Biliary; Biological Assay; Boxing; Cell Line; Cell Lineage; Cell Therapy; Cell Transplants; Cell fusion; Cells; Data; Development; Diploidy; Disease; Drug toxicity; Embryo; Epithelial Cells; Fibroblasts; Future; Gene Expression; Gene Expression Profile; Generations; Genes; Goals; Growth; Hepatic; Hepatitis B; Hepatitis C virus; Hepatocyte; Homeobox; Homologous Gene; Human; In Vitro; Injury; Karyotype; Lentivirus Vector; Liver; Liver Dysfunction; Liver Stem Cell; Liver diseases; Liver parenchyma; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of liver; Malignant neoplasm of pancreas; Memory; Metabolic Diseases; Molecular Genetics; Molecular and Cellular Biology; Morphology; Mus; Neoplastic Cell Transformation; Octamer Transcription Factor-3; Organ Transplantation; Patients; Pharmacologic Substance; Phenotype; Population; Proliferating; Property; Protocols documentation; Regenerative Medicine; Replacement Therapy; Research; Residual state; SCID Mice; Signal Pathway; Site; Somatic Cell; Staging; Stem Cell Research; Stem cells; Stimulus; Subfamily lentivirinae; System; Teratoma; Testing; Therapeutic; Tissues; Transplantation; Work; Xenobiotics; c-myc Genes; cancer site; cell type; cholangiocyte; clinical application; comparative; disease mechanisms study; embryonic stem cell; fetal; gene replacement therapy; human embryonic stem cell; improved; induced pluripotent stem cell; interest; liver transplantation; mouse model; novel; oval cell; pathogen; pluripotency; progenitor; reconstitution; stem; stem cell niche; stem cell technology; success

We have established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate the diseased liver upon transplantation using a MUP-uPA/SCID mouse model of liver injury. The FACS-purified hepatic progenitorcells developed into mature hepatocytes without evidence of cell fusion and participated in rebuilding normal parenchyma with reconstitution of liver-specific zonal gradients of hepatic function. The ES cell-derived hepatocytes were responsive to normal growthregulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl4-induced liver injury. The transplanted cells also differentiated into biliary epithelial cells. These data demonstrate that a highly enriched population ofcommitted hepatocyte precursors can be generated from mouse ES cells in vitro for effective cell replacement therapy.In addition, we have now established a protocol for generating iPSCs from human fibroblasts by transduction with lentiviruses that independently expressed POU domain class 5 transcription factor 1 (OCT3/4) SRY-box containing gene 2, (SOX2),NANOG homeobox (NANOG), and Lin-28 homolog (LN28). These iPSCs recapitulate both the hepatocytic differentiation and morphological changes seen with the human ES cells. Recent studies suggested that induced pluripotent stem cells (iPSCs) retain a residual donor cell gene expression, which may impact their capacity to differentiate into cell of origin. We have recently addressed a contribution of a lineage stage-specific donor cell memory in modulating the functional properties of iPSCs. iPSCs were generated from hepatic lineage cells at an early (hepatoblast-derived, HB-iPSCs) and end stage (adult hepatocyte, AH-iPSCs) of hepatocyte differentiation as well as from mouse embryonic fibroblasts (MEFs-iPSCs) using a lentiviral vector encoding four pluripotency-inducing factors Oct4, Sox2, Klf4, and c-Myc. All resulting iPSC lines acquired iPSCs phenotype as judged by the accepted criteria including morphology, expression of pluripotency markers, silencing of transducing factors, capacity of multilineage differentiation in teratoma assay, and normal diploid karyotype. However, HB-iPSCs were more efficient in directed differentiation toward hepatocytic lineage as compared to AH-iPSCs, MEF-iPSCs, or mouse embryonic stem cells (mESCs). Extensive comparative transcriptome analyses of the early passage iPSCs, donor cells, and mESCs revealed that despite global similarities in gene expression patterns between generated iPSCs and mESCs, HB-iPSCs retained a transcriptional memory (seven upregulated and 17 downregulated genes) typical of the original cells. Continuous passaging of HB-iPSCs erased most of these differences including a superior capacity for hepatic redifferentiation. These results suggest that retention of lineage stage-specific donor memory in iPSCs may facilitate differentiation into donor cell type. The identified gene set may help to improve hepatic differentiation for therapeutic applications and contribute to the better understanding of liver development.

我们已经建立了一个有效的系统，从小鼠胚胎干细胞（ES）细胞分化，扩增和分离肝祖细胞，并评估其能力，重新移植后使用MUP-uPA/SCID小鼠模型的肝损伤的患病肝脏。流式细胞仪纯化的肝祖细胞发育为成熟的肝细胞，没有细胞融合的证据，并参与重建正常的肝实质，重建肝脏特异性的肝功能区域梯度。ES细胞衍生的肝细胞对正常的生长调节有反应，并且在CCl 4诱导的肝损伤的额外生长刺激后以与宿主肝细胞相同的速率增殖。移植的细胞还分化为胆管上皮细胞。此外，我们现在已经建立了一种通过用慢病毒转导从人成纤维细胞产生iPSC的方案，所述慢病毒独立地表达POU结构域5类转录因子1（OCT 3/4）SRY盒，所述SRY盒包含基因2（SOX 2），NANOG同源盒（NANOG），和Lin-28同系物（LN 28）。这些iPSC概括了人类ES细胞所见的肝细胞分化和形态学变化。最近的研究表明，诱导多能干细胞（iPSC）保留了残余的供体细胞基因表达，这可能会影响其分化为原始细胞的能力。我们最近已经解决了谱系阶段特异性供体细胞记忆在调节iPSC的功能特性中的贡献。使用编码四种多能诱导因子Oct 4、Sox 2、Klf 4和c-Myc的慢病毒载体，从肝细胞分化的早期（成肝细胞衍生的，HB-iPSC）和末期（成体肝细胞，AH-iPSC）的肝谱系细胞以及从小鼠胚胎成纤维细胞（MEFs-iPSC）产生iPSC。所有得到的iPSC系获得iPSC表型，如通过公认的标准判断的，所述标准包括形态学、多能性标志物的表达、转导因子的沉默、畸胎瘤测定中的多谱系分化能力和正常二倍体核型。然而，与AH-iPSC、MEF-iPSC或小鼠胚胎干细胞（mESC）相比，HB-iPSC在向肝细胞谱系定向分化方面更有效。对早期传代iPSC、供体细胞和mESC的广泛比较转录组分析显示，尽管所产生的iPSC和mESC之间的基因表达模式具有全局相似性，但HB-iPSC保留了原始细胞典型的转录记忆（7个上调和17个下调基因）。HB-iPSC的连续传代消除了大部分这些差异，包括肝再分化的上级能力。这些结果表明，iPSC中谱系阶段特异性供体记忆的保留可以促进分化为供体细胞类型。鉴定的基因集可能有助于改善肝分化的治疗应用，并有助于更好地了解肝脏发育。

项目成果

Contribution of hepatic lineage stage-specific donor memory to the differential potential of induced mouse pluripotent stem cells.

• DOI: 10.1002/stem.1074
• 发表时间: 2012-05
• 期刊: STEM CELLS
• 影响因子: 5.2
• 作者: Lee, Seung Bum;Seo, Daekwan;Choi, Dongho;Park, Kye-Yoon;Holczbauer, Agnes;Marquardt, Jens U.;Conner, Elizabeth A.;Factor, Valentina M.;Thorgeirsson, Snorri S.
• 通讯作者: Thorgeirsson, Snorri S.