The selection of the appropriate extracellular matrix is critical for hESC self-renewal and proliferation,13, 22
and here we show that the ECMP composition also potently influences hESC differentiation to DE. By systematically screening hundreds of ECMP combinations, we identified two ECMPs, FN, and VTN, which significantly improve the efficiency of hESC differentiation to DE, thereby overcoming the need for poorly defined and non-human biological components, such as MGEL, in the manipulation of hESCs.
While certain studies have explored the role of physical properties of the microenvironment, including three-dimensional culture25
and substrate rigidity,26
we focused our analysis on the role of the ECM in differentiation and found that changes in the composition of the ECM profoundly affected the differentiation of hESCs to endoderm. It is particularly important to note that the effects of the growth factors inducing DE (Wnt3a and Activin A) are significantly influenced by ECMP composition. Our results suggest that appropriately defining the ECMP substrate in addition to the soluble signaling molecule environment is critical for improving the differentiation of hESCs to specific lineages.
The differentiation of hESCs to DE resembles that of primitive streak formation and gastrulation where cells invaginate and generate mesendodermal cell populations. These movements of epiblast cells require several growth factor signaling pathways, as well as an ECM. In this process the ECM does not merely function as a scaffold through which cells migrate. Rather, as determined by computational and optical methods, migrating cells move in concert with the ECM with little cellular movement relative to the ECM.27
This study supports the notion that the ECM has a more active role in development than previously appreciated and further underscores the importance of performing screens to identify optimal ECM compositions that promote specific developmental processes.
Consistent with our identification of FN and VTN as critical components that promote endodermal differentiation in cell culture, several studies in model organisms have provided compelling evidence that these ECM components are critical constituents of the microenvironment guiding the processes of primitive streak formation and gastrulation. For example, injection of agents that disrupt integrin-FN interactions, such as RGDS peptides or antibodies and Fab' fragments directed against FN, into chick embryos perturb gastrulation movements.28
Such microinjection experiments in frog embryos have led to similar observations.29
Mice lacking FN-binding integrins die early in development and fail to extend the anterior–posterior axis.30, 31, 32
Earlier defects, such as during gastrulation, are likely not uncovered due to rescue by maternally contributed FN message and protein. Together, countless studies in a variety of model organisms support the concept that the ECM, and specifically FN, has an important and instructive function in early embryogenesis. However, it should be stressed that in our studies only the initial matrix compositions are specified. Cells exposed to these ECMPs remodel the underlying matrix and begin secreting their own ECMPs. Even so, the observed cellular responses are a result of their exposure to the initial composition of the ECM.
Previous studies demonstrated that undifferentiated hESCs express a variety of integrins, including integrins α
1, 2, 3, 5, 6, 7, 11, E, and V, and β
1, 2, 3, and 5.33, 34, 35
We extended these studies by examining integrin gene expression in undifferentiated hESCs and hESC differentiated to each of the three primitive germ layers—endoderm, mesoderm, and ectoderm. This analysis revealed a specific integrin ‘signature' that was unique to each of these cell populations. Specifically, we found that ITGA5
gene expression was highly upregulated and ITGA6
expression was significantly downregulated in the endodermal lineage. Treatment with specific integrin blocking antibodies revealed that blocking ITGA5 impaired adhesion to FN, blocking ITGAV and ITGB5 reduced the binding to VTN, and blocking ITGA6 inhibited binding to LN.22
Furthermore, ITGA6 binding to LN has been implicated as having a critical role in the self-renewal and maintenance of pluripotent hESCs.36
In this study, we implemented an inducible shRNA system to demonstrate that knockdown of ITGA5 and ITGAV impaired endoderm formation. During development, integrin switching, rapid changes in the proportions of specific integrin subunits expressed at the cell surface, has been implicated as a mechanism that regulates cell differentiation.37, 38
Together our results suggest a possible mechanism in which hESCs differentiating to DE undergo an integrin switch from an ITGA6 signature which favors binding LN, and thereby maintenance of pluripotency, to an ITGA5 and ITGAV signature, which allows for interaction with FN and VTN and subsequent differentiation to DE.
Mouse models have been used extensively to interrogate integrin expression and functionality during embryonic development.37, 39, 40
Interestingly, mouse embryos stained for different integrin subunits at E6.5 revealed that Itga5 expression was mainly restricted to endoderm,41
which is consistent with the ITGA5 expression patterns that we identified in hESC-derived DE. Furthermore, Itga5 and ItgaV are widely expressed during development of many organs of endodermal origin, such as the pancreas, liver, and lungs.42, 43, 44
Knockout of Itga5 or ItgaV resulted in embryonic lethality,32, 45
while tissue specific deletion of Itga5 or ItgaV resulted in vasculature and neuronal defects.18, 32, 45, 46
Therefore, the novel findings presented here, which demonstrate knockdown of ITGA5 and ITGAV in hESCs impaired endoderm formation in hESCs, suggests that similar integrin knockdown strategies in hESCs can be used to interrogate the function of various integrin-ECMP interactions during the earliest stages of human development.
Current hESC differentiation protocols are insufficient in creating pure cell populations, which are required for understanding human development and creating disease relevant models. Therefore, developing sorting strategies for flow cytometry-based isolation of highly pure populations of cells from differentiating hESC cultures is of particular interest.47, 48, 49, 50
By investigating the role of integrin-ECMP interactions in hESC differentiation to DE, we identified a panel of novel surface integrins, ITGA5 (CD49e) and ITGAV (CD51), that allow for the FACS-based isolation of endodermal cells. In the future, similar integrin ‘signatures' could be developed that would permit the isolation of lineage committed cells from mixed differentiated hESC cultures.