Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation

Background To maximize the translational utility of human induced pluripotent stem cells (iPSCs), the ability to precisely modulate the differentiation of iPSCs to target phenotypes is critical. Although the effects of the physical cell niche on stem cell differentiation are well documented, current approaches to direct step-wise differentiation of iPSCs have been typically limited to the optimization of soluble factors. In this regard, we investigated how temporally varied substrate stiffness affects the step-wise differentiation of iPSCs towards various lineages/phenotypes. Methods Electrospun nanofibrous substrates with different reduced Young’s modulus were utilized to subject cells to different mechanical environments during the differentiation process towards representative phenotypes from each of three germ layer derivatives including motor neuron, pancreatic endoderm, and chondrocyte. Phenotype-specific markers of each lineage/stage were utilized to determine differentiation efficiency by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence imaging for gene and protein expression analysis, respectively. Results The results presented in this proof-of-concept study are the first to systematically demonstrate the significant role of the temporally varied mechanical microenvironment on the differentiation of stem cells. Our results demonstrate that the process of differentiation from pluripotent cells to functional end-phenotypes is mechanoresponsive in a lineage- and differentiation stage-specific manner. Conclusions Lineage/developmental stage-dependent optimization of electrospun substrate stiffness provides a unique opportunity to enhance differentiation efficiency of iPSCs for their facilitated therapeutic applications. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0667-2) contains supplementary material, which is available to authorized users.


Differentiation of iPSCs
To study the effects of electrospun substrate stiffness on the differentiation of iPSCs, defined developmental-stage protocols for motor neuron, pancreatic endoderm, and chondrocytes were utilized. The iPSCs were pre-cultured on Geltrex ® -coated tissue culture plates (TCPS) according to the protocol recommendations (i.e., motor neuron differentiation required a three day proliferation culture period to reach near confluency prior to the induction of differentiation).
Initially, iPSCs were seeded at 70,000 cells/cm 2 on TCPS for propagation and induction of differentiation to stage 1, 2, or 3. Alternatively, to analyze the effects of substrate stiffness at stage 1 of differentiation, iPSCs were seeded directly onto electrospun substrates. The iPSCs which were differentiated on TCPS through stage 1 were seeded onto electrospun substrates for differentiation through stage 2. Similarly, cells differentiated on TCPS through stage 2 were seeded onto substrates for differentiation through stage 3. At the end of each differentiation stage, samples were either lysed for gene expression analysis or fixed for protein expression analysis.

Differentiation to motor neurons
To differentiate iPSCs towards a motor neuron lineage, a protocol by Chambers et al. was utilized to guide the cells through the ectodermal, neural progenitor, and motor neuron stages [1].

Differentiation to pancreatic endoderm
A protocol by Kroon et al. was utilized to differentiate iPSCs through mesendoderm, posterior foregut, and pancreatic endoderm stages [2]. Media was supplemented daily with stage-specific media and growth factors. For stage 1 differentiation the cells were cultured in RPMI Glutamax media (Gibco) supplemented with 100 ng/ml Activin A (PeproTech) and 25 ng/ml Wnt3a on day 1 and RPMI media with 0.2% vol/vol FBS supplemented with 100 ng/mL Activin A on days 2-4.
At the start of stage 2, the wells were briefly washed with PBS containing calcium and magnesium and media was exchanged to RPMI media with 2% vol/vol FBS supplemented with 25-50 ng/ml KGF on days 5-9. On days 10-12 the media was changed to DMEM with 1% vol/vol B27 supplemented with 0.25 µM KAAD-cyclopamine (EMD Millipore, CA), 2 µM retinoic acid, and 50 ng/ml noggin. Stage 3 was induced on days 13-16 by DMEM with 1% vol/vol B27 and no additional growth factors.

Gene and protein expression analysis
The cells were harvested at the end of each differentiation stage for total RNA extraction using an RNeasy Micro Kit (Qiagen, CA) following manufacturer instructions. Complementary DNA (cDNA) synthesis was performed using an iScript cDNA Synthesis Kit (Bio-Rad, CA).
Quantitative real-time PCR was utilized to detect stage/lineage specific markers using custom primers (Table S1). Gene expression levels were analyzed using the comparative threshold cycle (CT) method with GAPDH used as an endogenous control [4].
To analyze the protein expression of stage/lineage-specific markers, cells were fixed with 4% paraformaldehyde for 30 minutes followed by staining using standard protocols. The respective images), as previously described [5]. Briefly, the images were converted to an 8-bit image type and the threshold was adjusted to highlight the cell nuclei. To divide merged cells the image was processed using the Watershed option. The 'analyze particles' function was used to determine the cell count. If the clarity of the images taken was not sufficient to accurately obtain a cell count the nuclei or positive cells were counted manually. At least 10 images were analyzed from stage-specific stained samples and the data is presented as mean ± standard deviation.

Statistical analysis
All experiments were conducted with at least triplicate samples and data is represented as means ± standard error of means (SEM) unless otherwise noted. Statistical significance was determined by the one sample student T-test using SPSS (v.23.0) software. A 'p' value of 0.05 or less was considered statistically significant.

TABLE S1. Custom real-time polymerase chain reaction (PCR) primers.
Sequences of forward and reverse primers for lineage-specific genes.

Primer
Forward Reverse