Roles of Integrins in Human Induced Pluripotent Stem Cell Growth on Matrigel and Vitronectin

Abstract

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived, patient-specific pluripotent cells for use in cell-based regenerative therapies. However, current methods of cell culture are tedious and expensive, and the mechanisms underlying cell proliferation are not understood. In this study, we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion, survival, and proliferation. We show that iPSC lines generated using Oct-3/4, Sox-2, Nanog, and Lin-28 express a repertoire of integrins similar to that of hESCs, with prominent expression of subunits alpha5, alpha6, alphav, beta1, and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin, in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel, as shown by immunological blockade experiments. On vitronectin, the integrin alphavbeta5 was required for initial attachment, but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore, iPSCs cultured on vitronectin for 9 passages retained normal karyotype, pluripotency marker expression, and capacity to differentiate in vitro. These studies suggest that vitronectin, or derivatives thereof, might substitute for Matrigel in a more defined system for iPSC culture.

Synthetic Surfaces for Human Embryonic Stem Cell Culture

“…These combinations, designated in Figure 1 as small orange circles, comprise cell surface receptors for all major ECM components. It was also found, as discussed in the previous section, that attachment through either laminin or RGD binding integrins , (Rodin et al, 2010), (Wong et al, 2010), (Braam et al, 2008), (Rowland et al, 2010), (Yoon et al, 2010), (Yap et al, 2011) is sufficient to support undifferentiated proliferation of hECS in serum-free, chemically defined medium. Fig.…”

“…The orthologs of human integrins recognizing the RGD amino acid sequence and laminins can be traced back to primitive organisms, while other integrins must have evolved with increasing organism complexity. From such a perspective, it is not surprising that hESCs primarily express integrins recognising the RGD sequence and laminin (Braam et al, 2008), (Meng et al, 2010), (Rowland et al, 2010), (Prowse et al, 2011). Braam et al, evaluated integrin expression in three hESC lines (HES2, HUES1, and HESCNL3) using fluorescence-activated cell sorting analysis.…”

Human embryonic stem cells and microenvironment

“…Farklı integrinlerle etkileşim sonucu ortak bir hücre içi sinyal mekanizmasının ya da alternatif mekanizmaların devreye girerek pluripotent özelliğin uzun süreli kontrolünü sağladığı dü-şünülmektedir. Son dönemde yapılan çalışmalarda besleyici hücrelerle olan kültür sistemlerinde, etkin bir in vitro sistem oluşturulabilmesi için iEKH ile besleyici hücrelerde uygun integrinlerin bulunması gerektiği gösterilmiştir [77][78][79] . Bu yüzden, hücre-lerdeki integrin reseptörlerinin ve buna bağlı sinyal yollarının tanımlanması pluripotent özelliğin uzun süreli korunumu ve uygun in vitro kültür şartlarının oluşturulabilmesi için gereklidir.…”

Mechanobiology of Human Pluripotent Stem Cells

“…53 a V b 5 is required for initial attachment of hiPSCs to a vitronectin substrate, but once attachment is achieved, the proliferation of these cells is not affected by blocking just a V b 5 with antibodies. 54 The inhibition of these cells' proliferation necessitates the blocking of both a V b 5 and b 1 . 54,55 Similarly, a V b 3 , a 6 b 1 , and a 2 b 1 play a decisive role in the adhesion and proliferation of hESCs on Matrigel substrates.…”

“…54 The inhibition of these cells' proliferation necessitates the blocking of both a V b 5 and b 1 . 54,55 Similarly, a V b 3 , a 6 b 1 , and a 2 b 1 play a decisive role in the adhesion and proliferation of hESCs on Matrigel substrates. 56 Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of integrin expression in mESCs indicated that integrins a 5 b 1 , a v b 5 , a 6 b 1 , and a 9 b 1 are involved in the maintenance of pluripotency, indicating that there is significant overlap in patterns of integrin expression between mPSCs and hPSCs.…”