Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, SE1 9RT, UK.
University of California, Berkeley, 278 Stanley Hall, Berkeley, CA 94720-3220, USA; Department of Pharmaceutical Chemistry, University of California at San Francisco, 600 16th St, Rm N512E, UCSF Box 2280, San Francisco, CA 94158, USA.
Acta Biomater. 2022 Sep 15;150:265-276. doi: 10.1016/j.actbio.2022.07.054. Epub 2022 Aug 1.
The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or 'niche'. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo. We therefore developed experimental models to investigate the combined effects of cell-ECM adhesion and cell-cell contact. We utilized lipid-modified oligonucleotides to form clusters of keratinocytes which were subsequently placed in suspension to induce terminal differentiation. In this experimental model cell-cell contact had no effect on suspension-induced differentiation of keratinocytes. We next developed a high-throughput platform for robust geometrical confinement of keratinocytes to hexagonal ECM-coated islands permitting direct cell-cell contact between single cells. As in the case of circular islands, differentiation was stimulated on the smallest single hexagonal islands. However, the percentage of involucrin-positive cells on small bowtie islands was significantly lower than on single islands, demonstrating that cell-cell contact reduced differentiation in response to decreased substrate adhesion. None of the small bowtie islands contained two involucrin-positive cells. Rather, if one cell was involucrin-positive the other was involucrin-negative. This suggests that there is intrinsic asymmetry in the effect of cell-cell contact in decreasing differentiation. Thus, our reductionist approaches provide new insights into the effect of the niche on keratinocyte differentiation. STATEMENT OF SIGNIFICANCE: Stem cell behaviour is regulated by a combination of external signals, including the nature of the adhesive substrate and cell-cell interactions. An understanding of how different signals are integrated creates the possibility of developing new biomaterials to promote tissue regeneration and broaden our understanding of skin diseases such as eczema and psoriasis, in which stem cell proliferation and differentiation are perturbed. In this study we have applied two methods to engineer intercellular adhesion of human epidermal stem cells, one involving lipid-modified DNA and the other involving hexagonal micropatterns. We show that the effect of cell-cell adhesion depends on cell-substrate adhesion and uncover evidence that two cells in equivalent environments can nevertheless behave differently.
干细胞的更新和分化之间的平衡是由内在细胞控制和由微环境（或“生态位”）提供的外在因素之间的相互作用决定的。以前关于培养的人类表皮的研究利用悬浮培养和限制细胞扩散来研究单个角质形成细胞分化的调节。然而，角质形成细胞在体内通常附着于邻近细胞。因此，我们开发了实验模型来研究细胞-细胞外基质粘附和细胞-细胞接触的综合影响。我们利用脂质修饰的寡核苷酸形成角质形成细胞簇，然后将其置于悬浮液中以诱导终末分化。在这个实验模型中，细胞-细胞接触对悬浮诱导的角质形成细胞分化没有影响。接下来，我们开发了一种高通量平台，用于将角质形成细胞牢固地限制在六边形细胞外基质包被的岛屿上，从而允许单细胞之间的直接细胞-细胞接触。与圆形岛屿一样，在最小的单个六边形岛屿上刺激分化。然而，在小蝴蝶结形岛上，角蛋白阳性细胞的百分比明显低于单个岛屿，这表明细胞-细胞接触减少了对基底粘附减少的分化。没有一个小蝴蝶结岛包含两个角蛋白阳性细胞。相反，如果一个细胞是角蛋白阳性的，那么另一个细胞就是角蛋白阴性的。这表明细胞-细胞接触在减少分化方面存在内在的不对称性。因此，我们的简化方法为生态位对角质形成细胞分化的影响提供了新的见解。意义声明：干细胞行为受外部信号的组合调节，包括粘附基底的性质和细胞-细胞相互作用。理解不同信号如何整合为一体，为开发新的生物材料以促进组织再生和拓宽我们对湿疹和银屑病等皮肤疾病的理解创造了可能性，在这些疾病中，干细胞增殖和分化受到干扰。在这项研究中，我们应用了两种方法来工程化人类表皮干细胞的细胞间粘附，一种涉及脂质修饰的 DNA，另一种涉及六边形微图案。我们表明，细胞-细胞粘附的效果取决于细胞-基底粘附，并揭示了在相同环境中两个细胞仍然可以表现出不同的行为的证据。
