We have shown that ECM stiffness regulates early differentiation but not self-renewal of hPSCs. In particular, while hPSC morphology is mechanosensitive under growth conditions, since cell and colony areas increase with increasing ECM stiffness, pluripotency marker expression and cell proliferation are not affected by ECM stiffness. In contrast, early neurogenic differentiation into SOX1 positive neural ectoderm prior to neural patterning is strongly modulated by this biophysical input. After further neural patterning and maturation, this effect translates to higher percentages of total neurons as well as dopaminergic neurons.
The relative stiffness-insensitivity of hPSC pluripotency marker expression contrasts with the observed rescue of mESC pluripotency on soft ECMs in the absence of growth factors6a
. In fact, while Tra-1-60 expression did not decrease significantly for the MSC-iPS upon growth factor withdrawal as it did for the H1 cells (likely due to differences in basal mTeSR versus X-VIVO media conditions), neither culture survived past 6 days in the absence of growth factors regardless of substrate stiffness. These results suggest that continued investigations into ESCs derived from both species will be needed to develop a more complete picture of the nature of pluripotency. These species-dependent differences are consistent with past comparative observations between hPSCs and mESCs. For example, these two cell types have exhibited differing responses to another biophysical cue, cyclic strain, which was shown to inhibit human ESC8
but promote mouse ESC6b
differentiation. These contrasting mechanosensitive phenotypes may arise from a growing list of observed differences in the fundamental cell biology of human and mouse ESCs, including in developmental stage14
, transcription factor binding15
, pluripotency marker expression16
, nuclear receptor expression during differentiation17
, keratin expression18
, and growth factors and signaling pathways that maintain pluripotency19
. In the future it may be interesting to compare potential crosstalk between these numerous factors and candidate mechanotransductive signaling pathways5c, 20
to elucidate species-distinct mechanosensitive behaviors and to understand biomedically relevant, human-specific properties that could be harnessed for therapeutic application.
Our finding that hiPSCs, like adult NSCs5c, 12
, increase neurogenesis on softer substrates offers important implications for the future development of biomedical therapies. hiPSCs in particular hold promise for patient-specific cell replacement therapies since they may more effectively evade immune responses than allogeneic hESC grafts, as well as bypass potential ethical concerns and corresponding supply limitations of embryo-derived hESCs in some countries. hiPSCs did exhibit lower overall levels of neurogenesis compared to hESCs, potentially due to epigenetic memory of their mesenchymal origins21
. However, the fact that hiPSC neural differentiation was still mechanosensitive despite epigenetic and transcriptomic differences between hiPSCs and hESCs22
and significantly different methods of derivation2a, 9
suggests the the observed mechanosensitivity of neuralization may generalize to many different types of hPSCs.
We and others have previously shown that ECM stiffness can modulate adult neural stem cell (aNSC) differentiation into neurons, astrocytes, and oligodendrocytes12, 23
, and the use of hPSCs in this study allows investigation of progenitor cells representative of earlier developmental periods. Culturing hPSCs on soft ECMs that mimic the stiffness of neural tissue (~100–1500 Pa) promoted the generation of neurons as it did with aNSCs. However, in contrast to the alteration of neuronal lineage commitment observed for aNSCs, for hPSCs the stiffness effect was mediated by increasing the percentage of early (SOX1+) neural progenitors. Interestingly, exposure to soft ECMs for only 5 out of a total of 19 days was sufficient to observe the downstream increase in neurons. Implementation of this “stiffness pulse” strategy thus reveals that when
a signal is presented may be just as important as what
signal is presented. Given that mechanical properties can function during multiple stages of differentiation, from neural conversion of hPSCs to neuronal differentiation and maturation of aNSCs, stem cell differentiation protocols that rely primarily on soluble media conditions1b
could be further improved by designing an optimal and temporally dynamic biophysical microenvironment. Our findings can therefore be applied to engineer biomaterials scaffolds and bioreactors for human pluripotent stem cell differentiation.
Future work could investigate extension of these observations to non-neural lineages, as well as address potential mechanisms responsible for our observations. In our system, softer ECMs resulted in lower extents of cell and colony spreading but did not affect hPSC proliferation or colony formation. The resulting higher effective cell densities or packing may yield smaller and more condensed individual cells and nuclei, as well as impact both the quantity and quality of cell-cell contacts during subsequent cell differentiation. These factors may in turn invoke cell and nuclear size/shape mechanisms important in mesenchymal stem cell differentiation20c, 24
and/or cell packing/density effects found in hESC systems25
. It is interesting to note that, while Chambers and colleagues observed a bias in downstream neuronal subtype specification and neural patterning due to cell density/packing1a
, we instead observed an earlier bias in the generation of neural progenitors and early neural ectoderm, suggesting that different combinations of cell-cell contacts, cell density/packing, and/or perhaps cell/nuclear shape may play important roles throughout neurogenesis.
The shape of a cell has been shown to affect its mechanical properties20c
, and ECM stiffness may also directly modulate cellular mechanics, either or both of which could in turn affect neural differentiation. Due to low survival of single hPSCs and inefficient clonal growth26
, it is difficult to study the effects of ECM stiffness directly on single cells. However, intracellular probes of force generation and mechanical properties2728
, in conjunction with biochemical and genetic studies, may help elucidate mechanisms of mechanosensitive hPSC differentiation into neural lineages.