Functional Maturation of Induced Pluripotent Stem Cell Hepatocytes in Extracellular Matrix-A Comparative Analysis of Bioartificial Liver Microenvironments.
Functional Maturation of Induced Pluripotent Stem Cell Hepatocytes in Extracellular Matrix-A Comparative Analysis of Bioartificial Liver Microenvironments.
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DOI：
10.5966/sctm.2015-0235
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发表时间：
2016-09
影响因子：
6
通讯作者：
Wertheim JA
中科院分区：
文献类型：
--
作者：
Wang B;Jakus AE;Baptista PM;Soker S;Soto-Gutierrez A;Abecassis MM;Shah RN;Wertheim JA
比较了两种三维生物支架系统逆转诱导多能干细胞（iPSC）衍生肝细胞的主要局限性的能力。当在由细胞外基质（ECM）制成的支架内培养时，iPSC肝细胞的增殖和功能显著增强。与其他平台相比，这种ECM支架增强了iPSC肝细胞的表型成熟，这可能是由于其生物多样性的组成。诱导多能干细胞（iPSC）是一种新的诊断和潜在的治疗工具，用于疾病建模和评估药物的毒性。与完全发育的内源性细胞相比，源自iPSC的细胞谱系的常见限制是钝化的表型。我们研究了新型三维生物人工微环境对从iPSC分化并在无细胞肝源性细胞外基质（ECM）支架内生长的肝细胞样细胞的功能和成熟的影响。同时，我们还比较了生物绘图的聚-l-乳酸（PLLA）支架，其允许细胞在三维中生长并形成细胞-细胞接触，但单独注入I型胶原（PLLA-胶原支架）作为具有狭窄生物多样性的“解构”对照支架。在两种支架内培养的iPSC衍生的肝细胞保持活力，变得极化，并形成胆管样结构;然而，与在生物绘图PLLA胶原或Matrigel夹心对照培养物中生长的iPSC肝细胞相比，在ECM支架内生长的细胞具有显著更高的P450（CYP 2C 9、CYP 3A 4、CYP 1A 2）mRNA水平和代谢酶活性。此外，与PLLA-胶原支架或夹心培养相比，白蛋白合成速率接近原代冻存肝细胞的水平，胎儿特异性基因、甲胎蛋白和CYP 3A 7的转录较低。这些研究表明，两种无细胞三维培养系统增加了iPSC衍生肝细胞的功能。然而，与生物绘图的PLA-胶原支架相比，来自单独ECM的支架诱导了进一步的肝细胞成熟。这种效果可能是介导的ECM支架的复杂组合物相比，生物绘图的支架，这表明它们的效用，在体外肝细胞测定或药物发现。通过使用新技术开发三维（3D）支架，本研究表明，通过诱导多能干细胞（iPSC）技术衍生的肝细胞样细胞在3D细胞外基质支架上成熟，这是3D基质结构和支架生物学的结果。结果是改善的肝表型，具有增加的合成和催化效力，改善iPSC衍生的肝细胞的钝化表型，这是iPSC技术的关键限制。这些发现提供了对3D微环境对iPSC肝细胞的活力，增殖和功能的影响的见解，以产生更成熟的细胞群体用于细胞毒性研究和疾病建模。
The ability of two three-dimensional bioscaffold systems to reverse the primary limitations of induced pluripotent stem cell (iPSC)-derived hepatocytes was compared. Proliferation and function of iPSC hepatocytes were significantly enhanced when cultured within scaffolds made from extracellular matrix (ECM). This ECM scaffold enhanced phenotypic maturation of iPSC hepatocytes compared with other platforms, likely owing to its biologically diverse makeup. Induced pluripotent stem cells (iPSCs) are new diagnostic and potentially therapeutic tools to model disease and assess the toxicity of pharmaceutical medications. A common limitation of cell lineages derived from iPSCs is a blunted phenotype compared with fully developed, endogenous cells. We examined the influence of novel three-dimensional bioartificial microenvironments on function and maturation of hepatocyte-like cells differentiated from iPSCs and grown within an acellular, liver-derived extracellular matrix (ECM) scaffold. In parallel, we also compared a bioplotted poly-l-lactic acid (PLLA) scaffold that allows for cell growth in three dimensions and formation of cell-cell contacts but is infused with type I collagen (PLLA-collagen scaffold) alone as a “deconstructed” control scaffold with narrowed biological diversity. iPSC-derived hepatocytes cultured within both scaffolds remained viable, became polarized, and formed bile canaliculi-like structures; however, cells grown within ECM scaffolds had significantly higher P450 (CYP2C9, CYP3A4, CYP1A2) mRNA levels and metabolic enzyme activity compared with iPSC hepatocytes grown in either bioplotted PLLA collagen or Matrigel sandwich control culture. Additionally, the rate of albumin synthesis approached the level of primary cryopreserved hepatocytes with lower transcription of fetal-specific genes, α-fetoprotein and CYP3A7, compared with either PLLA-collagen scaffolds or sandwich culture. These studies show that two acellular, three-dimensional culture systems increase the function of iPSC-derived hepatocytes. However, scaffolds derived from ECM alone induced further hepatocyte maturation compared with bioplotted PLLA-collagen scaffolds. This effect is likely mediated by the complex composition of ECM scaffolds in contrast to bioplotted scaffolds, suggesting their utility for in vitro hepatocyte assays or drug discovery. Through the use of novel technology to develop three-dimensional (3D) scaffolds, the present study demonstrated that hepatocyte-like cells derived via induced pluripotent stem cell (iPSC) technology mature on 3D extracellular matrix scaffolds as a result of 3D matrix structure and scaffold biology. The result is an improved hepatic phenotype with increased synthetic and catalytic potency, an improvement on the blunted phenotype of iPSC-derived hepatocytes, a critical limitation of iPSC technology. These findings provide insight into the influence of 3D microenvironments on the viability, proliferation, and function of iPSC hepatocytes to yield a more mature population of cells for cell toxicity studies and disease modeling.