Leong Meng Fatt, Lu Hong Fang, Lim Tze Chiun, Du Chan, Ma Nina K L, Wan Andrew C A
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore.
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore.
Acta Biomater. 2016 Dec;46:266-277. doi: 10.1016/j.actbio.2016.09.032. Epub 2016 Sep 22.
The use of human induced pluripotent stem cells (hiPSCs) for clinical tissue engineering applications requires expansion and differentiation of the cells using defined, xeno-free substrates. The screening and selection of suitable synthetic substrates however, is tedious, as their performance relies on the inherent material properties. In the present work, we demonstrate an alternative concept for xeno-free expansion and differentiation of hiPSCs using synthetic substrates, which hinges on the structure-function relationship between electrospun polystyrene scaffolds (ESPS) and pluripotent stem cell growth. ESPS of differential porosity was obtained by fusing the fibers at different temperatures. The more porous, loosely fused scaffolds were found to efficiently trap the cells, leading to a large number of three-dimensional (3D) aggregates which were shown to be pluripotent colonies. Immunostaining, PCR analyses, in vitro differentiation and in vivo teratoma formation studies demonstrated that these hiPSC aggregates could be cultured for up to 10 consecutive passages (P10) with maintenance of pluripotency. Flow cytometry showed that more than 80% of the cell population stained positive for the pluripotent marker OCT4 at P1, P5 and P10. P10 cells could be differentiated to neuronal-like cells and cultured within the ESPS for up to 18months. Our results suggest the usefulness of a generic class of synthetic substrates, exemplified by ESPS, for 'trapped aggregate culture' of hiPSCs.
To realize the potential of human induced pluripotent stem cells (hiPSCs) in clinical medicine, robust, xeno-free substrates for expansion and differentiation of iPSCs are required. In the existing literature, synthetic materials have been reported that meet the requirement for non-xenogeneic substrates. However, the self-renewal and differentiation characteristics of hiPSCs are affected differently by the biocompatibility and physico-chemical properties of individual substrates. Although some rules based on chemical structure and substrate rigidity have been developed, most of these efforts are still empirical, and most synthetic substrates must still be rigorously screened for suitability. In this paper, we demonstrate an alternative concept for xeno-free expansion and differentiation of hiPSCs using synthetic substrates, which hinges on the structure-function relationship between electrospun polystyrene scaffolds (ESPS) and pluripotent stem cell growth. ESPS of differential porosity was obtained by fusing the fibers at different temperatures. The more porous, loosely fused scaffold was found to efficiently trap the cells, leading to a large number of three-dimensional (3D) aggregates. In the form of these trapped aggregates, we showed that hiPSCs could be cultured for up to 10 consecutive passages (P10) with maintenance of pluripotency, following which they could be differentiated to a chosen lineage. We believe that this novel, generic class of synthetic substrates that employs 'trapped aggregate culture' for expansion and differentiation of hiPSCs is an important conceptual advance, and would be of high interest to the readership of Acta Biomaterialia.
将人诱导多能干细胞（hiPSC）用于临床组织工程应用时，需要使用确定的无动物源底物对细胞进行扩增和分化。然而，筛选和选择合适的合成底物很繁琐，因为它们的性能依赖于固有的材料特性。在本研究中，我们展示了一种使用合成底物对hiPSC进行无动物源扩增和分化的替代概念，该概念取决于电纺聚苯乙烯支架（ESPS）与多能干细胞生长之间的结构-功能关系。通过在不同温度下融合纤维获得了不同孔隙率的ESPS。发现孔隙率更高、融合较松散的支架能有效地捕获细胞，形成大量三维（3D）聚集体，这些聚集体被证明是多能集落。免疫染色、PCR分析、体外分化和体内畸胎瘤形成研究表明，这些hiPSC聚集体可连续培养多达10代（P10）并保持多能性。流式细胞术显示，在第1代、第5代和第10代时，超过80%的细胞群体对多能性标志物OCT4染色呈阳性。第10代细胞可分化为神经元样细胞，并在ESPS中培养长达18个月。我们的结果表明，以ESPS为代表的一类通用合成底物对于hiPSC的“捕获聚集体培养”是有用的。
为了实现人诱导多能干细胞（hiPSC）在临床医学中的潜力，需要用于iPSC扩增和分化的强大、无动物源底物。在现有文献中，已报道了符合非动物源底物要求的合成材料。然而，hiPSC的自我更新和分化特性受到各个底物的生物相容性和物理化学性质的不同影响。尽管已经制定了一些基于化学结构和底物刚性的规则，但这些努力大多仍属经验性，并且大多数合成底物仍必须经过严格筛选以确定其适用性。在本文中，我们展示了一种使用合成底物对hiPSC进行无动物源扩增和分化的替代概念，该概念取决于电纺聚苯乙烯支架（ESPS）与多能干细胞生长之间的结构-功能关系。通过在不同温度下融合纤维获得了不同孔隙率的ESPS。发现孔隙率更高、融合较松散的支架能有效地捕获细胞，形成大量三维（3D）聚集体。以这些捕获聚集体的形式，我们表明hiPSC可连续培养多达10代（P10）并保持多能性，随后它们可分化为选定的谱系。我们相信，这种采用“捕获聚集体培养”进行hiPSC扩增和分化的新型通用合成底物是一项重要的概念性进展，将引起《生物材料学报》读者的高度关注。
